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bioRxiv2018

Transcriptional logic of cell fate specification and axon guidance in early born retinal neurons revealed by single-cell mRNA profiling

Retinal ganglion cells (RGC), together with cone photoreceptors, horizontal cells (HC) and amacrine cells (AC), are the first classes of neurons produced in the retina. Here we have profiled 5348 single retinal cells and provided a comprehensive transcriptomic atlas showing the broad diversity of the developing retina at the time when the four early-born cells are being produced. Our results show the transcriptional sequences that establish the hierarchical ordering of early cell fate specification in the retina. RGC maturation follows six waves of gene expression, giving new insight into the regulatory logic of RGC differentiation. Early-generated RGCs transcribe an increasing amount of guidance cues for young peripheral RGC axons that express the matching receptors. Finally, spatial signatures in sub-populations of RGCs allowed to define novel molecular markers that are spatially restricted during the development of the retina. Altogether this study is a valuable resource that identifies new players in mouse retinal development, shedding light on transcription factors sequence and guidance cues dynamics in space and time.

developmental biology↗

bioRxiv2018

PTBP2-dependent alternative splicing regulates protein transport and mitochondria morphology in post-meiotic germ cells.

The RNA binding protein PTBP2 (polypyrimidine tract binding protein 2) is a key regulator of tissue-specific alternative RNA splicing. In the testis, PTBP2 is expressed in meiotic and post-meiotic germ cells (spermatocytes and spermatids, respectively). In these cells, PTBP2 is required for proper alternative mRNA splicing for over 200 genes, disproportionately affecting genes encoding proteins involved in protein trafficking via transport vesicles. In this study, we used electron microscopy to test the hypothesis that protein trafficking is impaired in the absence of PTBP2, and to further investigate why spermatogenesis abruptly ceases in PTBP2-deficienct spermatids. Ultrastructural analysis shows that protein trafficking in spermatids is aberrant in the absence of PTBP2. Unexpectedly, we also found that mitochondria morphology and number are significantly altered in PTBP2-deficient spermatids, consistent with increased mitochondria fission. Furthermore, we show that genes with key roles in mitochondria dynamics and function are post-transcriptionally regulated by PTBP2 and in different stages of spermatogenesis. Collectively, the data provide ultrastructural evidence that alternative splicing regulation by PTBP2 during spermatogenesis is critical for proper regulation of protein trafficking and mitochondria morphology.

developmental biology↗

bioRxiv2018

Enhancer priming enables fast and sustained transcriptional responses to Notch signaling

Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2/MCP system to visualize nascent transcripts in single cells in Drosophila embryos we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.

developmental biology↗

bioRxiv2018

Ribosomal DNA and the rDNA-binding protein Indra mediate non-random sister chromatid segregation in Drosophila male germline stem cells

Although considered to be exact copies of each other, sister chromatids can segregate non-randomly in some cases. For example, sister chromatids of the X and Y chromosomes segregate non-randomly during asymmetric division of male germline stem cells (GSCs) in D. melanogaster. Here we identify that the ribosomal DNA (rDNA) loci, which are located on the X and Y chromosomes, and an rDNA-binding protein, Indra, are required for non-random sister chromatid segregation (NRSS). We provide the evidence that NRSS is a mechanism by which GSCs recover rDNA copy number, which occurs through unequal sister chromatid exchange, counteracting the spontaneous copy number loss that occurs during aging. Our study reveals an unexpected role for NRSS in maintaining germline immortality through maintenance of a vulnerable genomic element, rDNA. One Sentence SummaryrDNA copy number maintenance by non-random sister chromatid segregation contributes to germline immortality in Drosophila

developmental biology↗

bioRxiv2018

Mitofusin 1 is required for the oocyte-granulosa cell communication that regulates oogenesis

Mitochondrial function, largely regulated by the dynamics of this organelle, is inextricably linked to oocyte health. While the proteins that modulate mitochondrial fusion, Mitofusin 1 (MFN1) and 2 (MFN2), are required for embryogenesis, their role in oocyte development remains unclear. Here we show that the oocyte-specific deletion of Mfn1, but not Mfn2, results in a complete loss of oocyte growth and ovulation due to a block in folliculogenesis at the preantral-to-antral follicle transition. We pinpoint the loss of oocyte ovulation to disrupted oocyte-somatic cell communication - Mfn1-null oocytes are deficient for the production of the important somatic cell signaling factor GDF9. Unexpectedly, the double loss of Mfn1 and Mfn2 mitigates the effects on oocyte growth and ovulation, which is explained by a partial rescue of oocyte-somatic cell communication and folliculogenesis. Together, this work demonstrates that mitochondrial function influences communication of oocyte with follicular somatic cells and suggests that the balanced expression of modulators of mitochondrial dynamics is critical for proper oocyte development.

developmental biology↗

bioRxiv2018

Sphingosine-1-Phosphate (S1P) analogue Phyto-sphingosine-1-Phosphate (P1P) improves the in vitro maturation efficiency of porcine oocytes via regulation of oxidative stress and apoptosis

Phytosphingosine-1-Phosphate (P1P) is a signaling sphingolipid regulating various physiological activities. Yet, little is known of the effect of P1P in the context of reproduction. As such, we aimed to investigate the influence of P1P on oocyte maturation during porcine in vitro maturation (IVM). Here we report the expression of S1PR1-3 among P1P receptors (S1PR1-4) in cumulus cells and oocytes. When P1P was treated by concentrations 10 nM, 50 nM, 100 nM, and 1000 nM during IVM, Metaphase II rate was significantly increased in 1000 nM (=1 M) P1P treatment group. Maturation rate improvement by P1P supplementation was only observed in the presence of EGF. Oocytes under the influence of P1P decreased intracellular ROS levels yet did not show significant differences in GSH levels. In our molecular studies, P1P treatment up-regulated gene expressions involved in cumulus expansion (Has2 and EGF), antioxidant enzyme (SOD3 and Cat), and developmental competence (Oct4) while activating ERK1/2 and Akt signaling. P1P treatment also influenced oocyte survival by shifting the ratio of Bcl-2 to Bax, while inactivating JNK signaling. We further demonstrated that oocytes matured with P1P significantly displayed not only higher developmental competence (cleavage and blastocyst formation rate), but also greater blastocyst quality (total cell number and the ratio of apoptotic cells) when activated via parthenogenetic activation (PA) and in vitro fertilization (IVF). Despite low levels of endogenous P1P found in animals, exogenous P1P was able to influence animal reproduction as shown by increased porcine oocyte maturation as well as preimplantation embryo development.

developmental biology↗

bioRxiv2018

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution

Direct comparisons of human and non-human primate brain tissue have the potential to reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is largely inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. First, we systematically evaluated the fidelity of organoid models to primary human and macaque cortex, finding organoid models preserve gene regulatory networks related to cell types and developmental processes but exhibit increased metabolic stress. Second, we identified 261 genes differentially expressed in human compared to chimpanzee organoids and macaque cortex. Many of these genes overlap with human-specific segmental duplications and a subset suggest increased PI3K/AKT/mTOR activation in human outer radial glia. Together, our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

developmental biology↗

bioRxiv2018

Fibroblast growth factor receptors function redundantly during zebrafish embryonic development

Fibroblast growth factor (Fgf) signaling regulates many processes during development. In many cases, one tissue layer secretes an Fgf ligand that binds and activates an Fgf receptor (Fgfr) expressed by a neighboring tissue. Although many Fgf ligands have known requirements in development, less is known about the requirements for the receptors. We have generated null mutations in each of the five fgfr genes in zebrafish. Considering the many requirements for Fgf signaling throughout development and that null mutations in the mouse Fgfr1 and Fgfr2 genes are embryonic lethal, it was surprising that all zebrafish homozygous mutants are viable and fertile, with no discernable embryonic defect. Instead, we have discovered surprising complexity of the Fgf pathway, where multiple receptors are involved in coordinating developmental processes. For example, mutations in the ligand fgf8a cause loss of the midbrain-hindbrain boundary, whereas in the fgfr mutants, this phenotype is only seen in embryos that are triple mutant for fgfr1a;fgfr1b;fgfr2, but not in any single and double mutant combinations. We show that this apparent fgfr redundancy is also seen during the development of several other tissues, including posterior mesoderm, pectoral fins, viscerocranium, and neurocranium. These data therefore begin to define the Fgfrs that function with a particular Fgf ligand to regulate early development in zebrafish.\n\nSummary statementAnalysis of fibroblast growth factor receptor mutants in zebrafish shows that functional redundancy assures robustness of a core developmental signaling pathway.

developmental biology↗

bioRxiv2018

Makorin1 controls embryonic patterning by alleviating Bruno-mediated repression of oskar translation

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning and germ cell specification. Here, we show that Mkrn1 is required for translational activation of oskar, whose product is critical for axis specification and germ plasm assembly. We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds osk 3 UTR in a region adjacent to A-rich sequences. This binding site also overlaps with Bruno (Bru) responsive elements (BREs), which regulate osk translation. We observe increased association of the translational repressor Bru with osk mRNA upon depletion of Mkrn1, implying that the two proteins compete for osk binding. Consistently, reducing Bru dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation via displacing Bru from its 3 UTR.

developmental biology↗

bioRxiv2018

Measurement of junctional tension in epithelial cells at the onset of primitive streak formation in the chick embryo via non-destructive optical manipulation

Oriented cell intercalations and cell shape changes are key determinants of large-scale epithelial cell sheet deformations occurring during gastrulation in many organisms. In several cases directional intercalation and cell shape changes have been shown to be associated with a planar cell polarity in the organisation of the actinmyosin cytoskeleton of epithelial cells. This polarised cytoskeletal organisation has been postulated to reflect the directional tension necessary to drive and orient directional cell intercalations. We have now further characterised and applied a recently introduced non-destructive optical manipulation technique to measure the tension in individual cell junctions in the epiblast of chick embryos in the early stages of primitive streak formation. We have measured junctional tension as a function of position and orientation. Junctional tension of mesendoderm cells, the tissue that drives the formation of the streak, is higher than tension of junctions of cells in other parts of the epiblast. Furthermore, in the mesendoderm junctional tension is higher in the direction of intercalation. The data are fitted best with a Maxwell model and we find that both junctional tension and relaxation time are dependent on myosin activity.

developmental biology↗

bioRxiv2018

Diversification of retinoblastoma protein function associated with cis and trans adaptations

Retinoblastoma proteins are eukaryotic transcriptional co-repressors that play central roles in cell cycle control, among other functions. Although most metazoan genomes encode a single retinoblastoma protein, gene duplications have occurred at least twice: in the vertebrate lineage, leading to three genes encoding Rb, p107, and p130, while separately in the Drosophila lineage an ancestral Rbf1 gene and a derived Rbf2 gene. Structurally, Rbf1 resembles p107 and p130 most closely, and mutation of the gene is lethal, while Rbf2 is more divergent, and is not essential for development. Rbf1 has been demonstrated to be a potent repressor of canonical cell-cycle promoters, unlike Rbf2. The retention of Rbf2 over 60 million years in the entire Drosophila lineage points to essential functions, however. We show here that Rbf2 regulates a broad set of cell growth control related genes, and can antagonize Rbf1 on specific sets of promoters. Rbf2 null mutants exhibit abnormal development of the female reproductive tract, with reduced egg laying, while heterozygous null mutants exhibit an increased rate of egg deposition, suggesting that the normal function of this protein is critical for optimal control of fertility. The structural alterations found in conserved regions of the Rbf2 gene suggest that this gene was sub- or neofunctionalized to develop specific regulatory specificity and activity. We define cis regulatory features of Rbf2 target genes that allow preferential repression by this protein, indicating that it is not merely a weaker version of the ancestral protein. The specialization of retinoblastoma function in Drosophila may reflect a parallel evolution found in vertebrates, and raises the possibility that cell growth control is equally important to cell cycle function for this conserved family of transcriptional corepressors.

developmental biology↗

bioRxiv2018

Nessys: a novel method for accurate nuclear segmentation in 3D

Methods for measuring the properties of individual cells within their native 3D environment will enable a deeper understanding of embryonic development, tissue regeneration, and tumorigenesis. However current methods for segmenting nuclei in 3D tissues are not designed for situations where nuclei are densely packed, non-spherical, heterogeneous in shape, size, or texture, all of which are true of many embryonic and adult tissue types as well as in many cases for cells differentiating in culture.\n\nHere we overcome this bottleneck by devising a novel method based on labelling the nuclear envelope (NE) and automatically distinguishing individual nuclei using a tree structured ridge tracing method followed by shape ranking according to a trained classifier. The method is fast and makes it possible to process images that are larger than the computers memory. We consistently obtain accurate segmentation rates of >90% even for challenging images such as mid-gestation embryos or 3D cultures. We provide a 3D editor and inspector for the manual curation of the segmentation results as well as a program to assess the accuracy of the segmentation.\n\nWe have also generated a live reporter of the NE that can be used to track live cells in three dimensions over time. We use this to monitor the history of cell interactions and occurrences of neighbour exchange within cultures of pluripotent cells during differentiation.\n\nWe provide these tools in an open-access user-friendly format.

developmental biology↗

bioRxiv2018

Genetic approaches in mice demonstrate that neuro-mesodermal progenitors express T/Brachyury but not Sox2

Neural tube and somites have long been thought to derive from separate germ layers: the ectoderm and mesoderm. This concept was challenged by the discovery of neuro-mesodermal progenitors, a bi-potent cell population that gives rise to both spinal neural tube and somites. In line with their proposed potency, these cells are considered to co-express the neural marker Sox2 and the mesodermal marker T/Brachyury. We performed genetic lineage tracing in mouse embryos and confirmed that T-expressing cells give rise to both neural tube and mesoderm. Surprisingly, however, Sox2-expressing cell derivatives colonise only the neural tube after embryonic day 8.5. Deletion of Sox2 in T-expressing cells was compatible with an otherwise normal neural tube and paraxial mesoderm. Moreover, Sox2 expression is absent from the chordoneural hinge, where neuro-mesodermal progenitors are located. Our findings demonstrate that neuro-mesodermal progenitors express T but not Sox2, suggesting the need for re-evaluation of the neuro-mesodermal progenitor hypothesis.

developmental biology↗

bioRxiv2018

The Caenorhabditis elegans HAM-1 protein modifies G protein signaling and membrane extension to reverse the polarity of asymmetric cell division

Asymmetric divisions often produce daughter cells that differ in both fate and size. The Caenorhabditis elegans HAM-1 protein regulates both daughter cell fate and daughter cell size asymmetry (DCSA) in a subset of asymmetric divisions. Here we focus on the divisions of the Q.a and Q.p neuroblasts, which use distinct mechanisms to divide with opposite polarity. Q.a divides by a ham-1-dependent, spindle-independent, myosin-dependent mechanism to produce a smaller anterior daughter that dies, whereas Q.p divides by a ham-1-independent, spindle-dependent, myosin-independent mechanism to produce a smaller posterior daughter that dies. Despite these differences, we found that membrane extension at the posterior of Q.a and at the anterior of Q.p promoted DCSA in these cells by a Wiscott-Aldrich protein (WASp)-dependent mechanism and that in ham-1 mutant Q.a divisions, the polarity of this extension was reversed. In addition, the spindle moved posteriorly during the Q.a division in a ham-1 mutant, a phenotype normally exhibited by Q.p. We found that this spindle movement in wild-type Q.p divisions required Ga proteins that promote spindle movement in other asymmetric divisions, and GPR-1, a protein involved in linking G proteins to microtubule asters, localized to the posterior cortex of Q.p. Genetic interactions suggest that ham-1 mutant Q.a divisions also require Ga proteins function to divide with a reversed polarity. The transformation of Q.a to Q.p-like polarity in the ham-1 mutant, however, appeared incomplete: ham-1 loss did not alter the asymmetric localization of the non-muscle myosin NMY-2 to the anterior cortex of Q.a. A GFP tagged ham-1 transgene revealed that Q.a but not Q.p expressed ham-1. Finally, we show that HAM-1 has both cortical and nuclear functions in Q,a DCSA. We propose a model where HAM-1 modifies a default Q.p-type polarity by localizing WASp function to the posterior Q.a membrane and by interfering with G-protein mediated spindle movement.\n\nAuthor SummaryOne way that animals produce different cell types is by asymmetric cell division, where a cell divides to produce daughter cells that differ in fate. Much is known about the mechanisms that polarize dividing cells to generate daughters that differ in fate. Some asymmetric cell divisions also result in daughters that differ in size, and the mechanisms that regulate how cells generate an asymmetric cleavage plane are poorly understood. In neural progenitors of the nematode Caenorhabditis elegans, two distinct mechanisms generate daughter cells of different size. One type requires movement of the mitotic spindle, which then defines the plane of the cell division. The other is spindle-independent. Here, we study two cells that divide with opposite polarities using these two mechanisms. We find that in the absence of the protein HAM-1, which has been reported to regulate gene transcription, the cell that normally divides using a spindle-independent mechanism now divides with a reversed polarity using a spindle-dependent mechanism. Our findings suggest that HAM-1 plays a key role in defining the mechanism by which a progenitor divides to produce daughter cells of different sizes and that both localization to the cell periphery and nucleus are important for its function.

developmental biology↗

bioRxiv2018

A Regulatory Loop between the Retinoid-Related Orphan Nuclear Receptor NHR-23 and let-7 family microRNAs Modulates the C. elegans Molting Cycle

Animal development requires coordination among cyclic processes, sequential cell fate specifications, and once-a-lifetime morphogenic events, but the underlying mechanisms are not well understood. C. elegans undergo four molts at regular 8-10 h intervals. The pace of the cycle is governed by PERIOD/lin-42 and other as-yet unknown factors. Cessation of the cycle in young adults is controlled by the let-7 family of microRNAs and downstream transcription factors in the heterochronic pathway. Here, we characterize a negative feedback loop between NHR-23, the worm homolog of mammalian retinoid-related orphan receptors (RORs), and the let-7 family of microRNAs that regulates both the frequency and finite number of molts. The molting cycle is decelerated in nhr-23 knockdowns, accelerated in let-7(-) mutants, and similar to wild type animals in let-7(-) nhr-23(-) double mutants. NHR-23 binds response elements (ROREs) in the let-7 promoter and activates transcription. In turn, let-7 dampens nhr-23 expression across development via a complementary let-7 binding site (LCS) in the nhr-23 3' UTR. The molecular interactions between NHR-23 and let-7 hold true for other let-7 family microRNAs. Either derepression of nhr-23 transcripts by LCS deletion or high gene dosage of nhr-23 leads to protracted behavioral quiescence and extra molts in adults. NHR-23 and let-7 also co-regulate scores of genes required for execution of the molts, including lin-42. In addition, ROREs and LCSs isolated from mammalian ROR and let-7 genes function in C. elegans, suggesting conservation of this feedback mechanism. We propose that this feedback loop unites the molting timer and the heterochronic gene regulatory network possibly by functioning as a cycle counter.

developmental biology↗

bioRxiv2018

The role of Selenoprotein N in the differentiation of erythroid progenitors during stress erythropoiesis

Low serum Se is independently associated with anemia in elderly population, dialysis patients, sickle-cells patients, and hypothyroidism patients. Previous work from our laboratory showed that dietary Se deficiency in mice showed mild anemia indicating activation of stress-erythropoietic mechanisms. Unlike steady state erythropoiesis that is primarily responsible for homeostasis to produce new erythrocytes at a constant rate, stress erythropoiesis predominates when the bone marrow cannot generate sufficient erythrocytes. During such a process, short-term reconstituting hematopoietic stem cells (CD34+Kit+Sca1+Linneg) migrate to the spleen leading to the proliferation and differentiation of stress-erythroid progenitors (SEPs). These cells lead to stress burst forming unit-erythroid cells (BFU-E) followed by terminal differentiation to erythrocytes. Recent studies demonstrate deficits in selenoproteins block the expansion and development of stress BFU-E with defects in terminal differentiation. Analysis of selenoprotein expression showed that selenoprotein W (SelenoW) was highly expressed in developing SEPs. CRISPR-Cas9 knockout of SelenoW blocked the proliferation of immature SEPs in murine and human stress erythropoiesis cultures demonstrating a central role for SelenoW in stress erythropoiesis. Using the two-culture system to generate SEPs, selenoprotein N (SelenoN) expression increased as the progenitors transition from self-renewing \"stem cell like\" progenitors to form committed erythroid progenitors. SelenoN-/- mice showed significantly slower erythroid recovery following phenylhydrazine (PHZ)-induced acute hemolytic anemia. As in the muscle satellite cells where SelenoN regulates cellular Ca2+ signaling, SelenoN may also regulate Ca2+ signaling in SEPs to modulate pathways of differentiation. In summary, these data suggest that multiple selenoproteins, including SelenoN and SelenoW, coordinately regulate stress erythropoiesis.

developmental biology↗

bioRxiv2018

Prediction and control of symmetry breaking in embryoid bodies by environment and signal integration

During early embryogenesis, mechanical signals, localized biochemical signals and neighboring cell layers interaction coordinate around anteroposterior axis determination and symmetry breaking. Deciphering their relative roles, which are hard to tease apart in vivo, will enhance our understanding of how these processes are driven. In recent years, in vitro 3D models of early mammalian development, such as embryoid bodies (EBs) and gastruloids, were successful in mimicking various aspects of the early embryo, providing high throughput accessible systems for studying the basic rules shaping cell fate and morphology during embryogenesis. Using Brachyury (Bry), a primitive streak and mesendoderm marker in EBs, we study how contact, biochemical and neighboring cell cues affect the positioning of a primitive streak-like locus, determining the AP axis. We show that a Bry-competent layer must be formed in the EB before Bry expression initiates, and that Bry onset locus selection depends on contact points of the EB with its surrounding. We can maneuver Bry onset to occur at a specific locus, a few loci, or in an isotropic peripheral pattern. By spatially separating contact and biochemical signal sources, we show these two modalities can be integrated by the EB to generate a single Bry locus. Finally, we show Foxa2+ cells are predictive of the future location of Bry onset, demonstrating an earlier symmetry-breaking event. By delineating the temporal signaling pathway dependencies of Bry and Foxa2, we were able to selectively abolish either, or spatially decouple the two cell types during EB differentiation. These findings demonstrate multiple inputs integration during an early developmental process, and may prove valuable in directing in vitro differentiation.

developmental biology↗

bioRxiv2018

miRNAs, target genes expression and morphological analysis on the heart in gestational protein-restricted offspring

BACKGROUNDGestational protein restriction was associated with low birth weight, hypertension and higher prevalence of cardiac disorders in adults. Several mechanisms, including epigenetics, could be related with the cardiovascular phenotype on protein-restricted offspring. Thus, we investigated the morphological cardiac effects of gestational protein restriction and left ventricle miRNAs and target genes expression pattern in both 12-day and 16-week old gestational protein-restricted male offspring.\n\nMETHODSPregnant Wistar rats were allocated into two groups, according to protein supply during pregnancy: NP (normal protein diet-17%) or LP (low protein diet - 6%). The study evaluates the effects of maternal protein restriction on food consumption and body weight of both pregnant dams and offspring, systolic blood pressure in 16-wk old offspring and on cardiac morphometric and molecular parameters in both 12-d and 16-wk old offspring.\n\nRESULTSDams on the gestational protein-restricted diet had lower body weight gain and higher food intake. Gestational protein-restricted offspring had low birth weight, followed by rapidly body weight recovery, hypertension, and increased myocytes crosssectional area and collagen fraction at 16-week old age. At 12-days old, miR-184, miR-192, miR-376c, miR-380-3p, miR-380-5p, miR-451, and miR-582-3p had increased expression, and miR-547 and miR-743 a had decreased expression in the gestational protein-restricted left ventricle. At 16-week old, let-7b, miR-125a-3p, miR-142-3p, miR-182 and miR-188-5p had increased expression and let-7g, miR-107, miR-127, miR-181a, miR-181c, miR-184, miR-324-5p, miR-383, miR-423-5p and miR-484 had decreased expression in gestational protein-restricted left ventricle. Target predicted gene expression analysis shown higher expression of Dnmt3a, Oxct1, Rictor and Trps1 and lower expression of Bbs1 and Calml3 in 12-day old protein-restricted offspring. 16-week old protein-restricted offspring had higher expression of Adrbk1, Bbs1, Dnmt3a, Gpr22, Inppl1, and Oxct1 genes.\n\nCONCLUSIONGestational protein restriction leads to offspring low birth weight, increased systolic blood pressure and morphological heart alterations that could be related to early heart miRNA expression changes that perpetuate into adulthood and which are associated with the regulation of essential genes involved in cardiovascular development, heart morphology, function, and metabolism.

developmental biology↗