Transcriptional progression during meiotic prophase I reveals sex-specific features and X chromosome dynamics in human fetal female germline

PLoS genetics

2021 Sep 01

Fan, X;Moustakas, I;Torrens-Juaneda, V;Lei, Q;Hamer, G;Louwe, LA;Pilgram, GSK;Szuhai, K;Matorras, R;Eguizabal, C;Westerlaken, LV;Mei, H;Chuva de Sousa Lopes, SM;
PMID: 34499650 | DOI: 10.1371/journal.pgen.1009773

During gametogenesis in mammals, meiosis ensures the production of haploid gametes. The timing and length of meiosis to produce female and male gametes differ considerably. In contrast to males, meiotic prophase I in females initiates during development. Hence, the knowledge regarding progression through meiotic prophase I is mainly focused on human male spermatogenesis and female oocyte maturation during adulthood. Therefore, it remains unclear how the different stages of meiotic prophase I between human oogenesis and spermatogenesis compare. Analysis of single-cell transcriptomics data from human fetal germ cells (FGC) allowed us to identify the molecular signatures of female meiotic prophase I stages leptotene, zygotene, pachytene and diplotene. We have compared those between male and female germ cells in similar stages of meiotic prophase I and revealed conserved and specific features between sexes. We identified not only key players involved in the process of meiosis, but also highlighted the molecular components that could be responsible for changes in cellular morphology that occur during this developmental period, when the female FGC acquire their typical (sex-specific) oocyte shape as well as sex-differences in the regulation of DNA methylation. Analysis of X-linked expression between sexes during meiotic prophase I suggested a transient X-linked enrichment during female pachytene, that contrasts with the meiotic sex chromosome inactivation in males. Our study of the events that take place during meiotic prophase I provide a better understanding not only of female meiosis during development, but also highlights biomarkers that can be used to study infertility and offers insights in germline sex dimorphism in humans.

Gene expression of intracortical bone demonstrates loading-induced increases in Wnt1 and Ngf and inhibition of bone remodeling processes

Bone

2021 May 21

Harris, TL;Silva, MJ;
PMID: 34023542 | DOI: 10.1016/j.bone.2021.116019

Osteocytes are the primary mechanosensitive cells in bone. However, their location in mineralized matrix has limited the in vivo study of osteocytic genes induced by mechanical loading. Laser Capture Microdissection (LCM) allows isolation of intracortical bone (Intra-CB), enriched for osteocytes, from bone tissue for gene expression analysis. We used microarray to analyze gene expression from mouse tibial Intra-CB dissected using LCM 4 h after a single loading bout or after 5 days of loading. Osteocyte enrichment was supported by greater expression of Sost, Dmp1, Dkk1, and Mepe in Intra-CB regions vs. Mixed regions containing periosteum and muscle (fold-change (FC) = 3.4, 2.2, 5.1, 3.0, respectively). Over 150 differentially expressed genes (DEGs) due to loading (loaded vs. contralateral control) in Intra-CB were found on Day 1 and Day 5, but only 10 genes were differentially expressed on both days, including Ngf (Day 1 FC = 13.5, Day 5 FC = 11.1) and Wnt1 (Day 1 FC = 1.5, Day 5 FC = 5.1). The expression of Ngf and Wnt1 within Intra-CB was confirmed by in situ hybridization, and a significant increase in number of Wnt1 mRNA molecules occurred on day 1. We also found changes in extracellular matrix remodeling with Timp1 (FC = 3.1) increased on day 1 and MMP13 (FC = 0.3) decreased on day 5. Supporting this result, IHC for osteocytic MMP13 demonstrated a marginal decrease due to loading on day 5. Gene Ontology (GO) biological processes for loading DEGs indicated regulation of vasculature, neuronal and immune processes while cell-type specific gene lists suggested regulation of osteoclast, osteoblast, and endothelial related genes. In summary, microarray analysis of microdissected Intra-CB revealed differential regulation of Ngf, Wnt1, and MMP13 due to loading in osteocytes.

SLC20a1/PiT-1 is required for chorioallantoic placental morphogenesis

Vascular biology (Bristol, England)

2023 Feb 01

Correia-Branco, A;Mei, A;Pillai, SS;Jayaraman, N;Sharma, R;Paquette, AG;Neradugomma, NK;Benson, C;Chavkin, N;Mao, Q;Wallingford, MC;
PMID: 36795703 | DOI: 10.1530/VB-22-0018

The placenta mediates transport of nutrients, such as inorganic phosphate (Pi), between the maternal and fetal circulatory systems. The placenta itself also requires high levels of nutrient uptake as it develops, to provide critical support for fetal development. This study aimed to determine placental Pi transport mechanisms using in vitro and in vivo models. We observed that Pi (P33) uptake in BeWo cells is sodium-dependent, and that SLC20A1/Slc20a1 is the most highly expressed placental sodium-dependent transporter in mouse (microarray) and human cell line (RT-PCR) and term placenta (RNA-seq), supporting that normal growth and maintenance of the mouse and human placenta requires SLC20A1/Slc20a1. Slc20a1 wildtype (Slc20a1+/+) and knockout (Slc20a1-/-) mice were produced through timed intercrosses, and displayed yolk sac (YS) angiogenesis failure as expected at E10.5. E9.5 tissues were analyzed to test whether placental morphogenesis requires Slc20a1. At E9.5 the developing placenta was reduced in size in Slc20a1-/-. Multiple structural abnormalities were also observed in the Slc20a1-/- chorioallantois. We determined that MCT1+ cells were reduced in developing Slc20a1-/- placenta, confirming that Slc20a1 loss reduced trophoblast syncytiotrophoblast 1 (SynT-I) coverage. Next, we examined cell type-specific Slc20a1 expression and SynT molecular pathways in silico, and identified Notch/Wnt as a pathway of interest that regulates trophoblast differentiation. We further observed that specific trophoblast lineage express Notch/Wnt genes that associate with endothelial cell tip-and-stalk cell markers. In conclusion, our findings support that Slc20a1 mediates symport of Pi into SynT cells, providing critical support for their differentiation and angiogenic mimicry function at the developing maternal-fetal interface.

Fibroblast Growth Factor Regulates Olfactory Bulb Formation by Controlling Radial Glial Cell Development

SSRN Electronic Journal

2022 Nov 10

Ito, A;Imamura, F;
| DOI: 10.2139/ssrn.4267408

Fibroblast growth factor (FGF) signaling plays several important roles in the development of the central nervous system. During the mid-gestation stage, FGF receptors (FGFRs) are expressed in the ventricular zone of the telencephalon and regulate the proliferation and neuronal differentiation of radial glial cells (RGCs). Inhibition of FGFR signaling at this stage results in abnormal brain formation, particularly loss of FGFR1 signaling causes hypoplasia of the olfactory bulb (OB). However, how FGFR1 signaling regulates OB formation is not fully understood. In this study, we inhibited FGFR1 signaling specifically in the anterior telencephalon, where OBs develop, and examined its effects on the development of RGCs in the OB (OB RGCs) and OB formation. The results suggest that inhibition of FGFR1 signaling causes a shift in the state of OB RGCs from proliferation to neuronal differentiation, resulting in an insufficient number of OB projection neurons. Furthermore, activation of Notch signaling, which maintains the self-renewal state of OB RGCs, partially rescued the early abnormal OB formation caused by inhibition of FGFR1 signaling. In contrast, inhibition of FGFR1 signaling in lateral ganglionic eminence did not affect the production of OB interneurons or OB formation. Moreover, the early abnormal OB formation induced by inhibition of FGFR1 signaling could be rescued by overactivation of Notch signaling, which maintains the proliferative state of radial glial cells. These results suggest that FGFR1 signaling regulates normal OB formation by controlling OB RGCs to produce a normal number of OB projection neurons.

Retinal ganglion cell expression of cytokine enhances occupancy of NG2 cell-derived astrocytes at the nerve injury site: Implication for axon regeneration

Experimental neurology

2022 Jun 20

Ribeiro, M;Ayupe, AC;Beckedorff, FC;Levay, K;Rodriguez, S;Tsoulfas, P;Lee, JK;Nascimento-Dos-Santos, G;Park, KK;
PMID: 35738417 | DOI: 10.1016/j.expneurol.2022.114147

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

Orphan G-Protein Coupled Receptor GPRC5B Is Critical for Lymphatic Development

International journal of molecular sciences

2022 May 20

Xu, W;Nelson-Maney, NP;Bálint, L;Kwon, HB;Davis, RB;Dy, DCM;Dunleavey, JM;St Croix, B;Caron, KM;
PMID: 35628521 | DOI: 10.3390/ijms23105712

Numerous studies have focused on the molecular signaling pathways that govern the development and growth of lymphatics in the hopes of elucidating promising druggable targets. G protein-coupled receptors (GPCRs) are currently the largest family of membrane receptors targeted by FDA-approved drugs, but there remain many unexplored receptors, including orphan GPCRs with no known biological ligand or physiological function. Thus, we sought to illuminate the cadre of GPCRs expressed at high levels in lymphatic endothelial cells and identified four orphan receptors: GPRC5B, AGDRF5/GPR116, FZD8 and GPR61. Compared to blood endothelial cells, GPRC5B is the most abundant GPCR expressed in cultured human lymphatic endothelial cells (LECs), and in situ RNAscope shows high mRNA levels in lymphatics of mice. Using genetic engineering approaches in both zebrafish and mice, we characterized the function of GPRC5B in lymphatic development. Morphant gprc5b zebrafish exhibited failure of thoracic duct formation, and Gprc5b-/- mice suffered from embryonic hydrops fetalis and hemorrhage associated with subcutaneous edema and blood-filled lymphatic vessels. Compared to Gprc5+/+ littermate controls, Gprc5b-/- embryos exhibited attenuated developmental lymphangiogenesis. During the postnatal period, ~30% of Gprc5b-/- mice were growth-restricted or died prior to weaning, with associated attenuation of postnatal cardiac lymphatic growth. In cultured human primary LECs, expression of GPRC5B is required to maintain cell proliferation and viability. Collectively, we identify a novel role for the lymphatic-enriched orphan GPRC5B receptor in lymphangiogenesis of fish, mice and human cells. Elucidating the roles of orphan GPCRs in lymphatics provides new avenues for discovery of druggable targets to treat lymphatic-related conditions such as lymphedema and cancer.

Kidney-Specific KO of the Circadian Clock Protein PER1 Alters Renal Sodium Handling, Aldosterone Levels, and Kidney/Adrenal Gene Expression

American journal of physiology. Renal physiology

2022 Feb 07

Douma, LG;Costello, HM;Crislip, GR;Cheng, KY;Lynch, IJ;Juffre, A;Barral, D;Masten, SH;Roig, E;Beguiristain, K;Li, W;Bratanatawira, P;Wingo, CS;Gumz, ML;
PMID: 35129370 | DOI: 10.1152/ajprenal.00385.2021

PER1 is a circadian clock transcription factor that is regulated by aldosterone, a hormone that increases blood volume and sodium retention to increase blood pressure. Male global Per1 knockout (KO) mice develop reduced night/day differences in sodium excretion in response to a high salt diet plus desoxycorticosterone pivalate treatment (HS+DOCP), a model of salt-sensitive hypertension. Additionally, global Per1 KO mice exhibit higher aldosterone levels on a normal salt diet. To determine the role of Per1 in the kidney, male kidney-specific Per1 KO (KS-Per1 KO) mice were generated using Ksp-cadherin Cre recombinase to remove exons 2-8 of Per1 in the distal nephron and collecting duct. Male KS-Per1 KO mice have increased sodium retention, but have normal diurnal differences in sodium excretion in response to HS+DOCP. The increased sodium retention is associated with altered expression of glucocorticoid and mineralocorticoid receptors, increased serum aldosterone, and increased medullary endothelin-1 compared to control (CNTL) mice. Adrenal gland gene expression analysis revealed that circadian clock and aldosterone synthesis genes have altered expression in the KS-Per1 KO mice compared to CNTL mice. These results emphasize the importance of the circadian clock, not only in maintaining rhythms of physiological functions but also for adaptability in response to environmental cues, such as HS+ DOCP, to maintain overall homeostasis. Given the prevalence of salt-sensitive hypertension in the general population, these findings have important implications for our understanding of how circadian clock proteins regulate homeostasis.

Secreted protease ADAMTS18 in development and disease

Gene

2023 Jan 09

Nie, J;Zhang, W;
PMID: 36632911 | DOI: 10.1016/j.gene.2023.147169

ADAMTS18 was identified in 2002 as a member of the ADAMTS family of 19 secreted Zinc-dependent metalloproteinases. Prior to 2016, ADAMTS18 was known as a candidate gene associated with a wide range of pathologies, particularly various malignancies and eye disorders. However, functions and substrates of ADAMTS18 in normal conditions were unknown. Since 2016, with the development of Adamts18 knockout models, many studies had been conducted on the Adamts18 gene in vivo. These studies revealed that ADAMTS18 is essential for the morphology and organogenesis of several epithelial organs (e.g., lung, kidney, breast, salivary glands, and lacrimal glands), vascular and neuronal systems, adipose tissue, and reproductive tracts. In this review, we describe the current understanding of ADAMTS18 and its substrates and regulators. Limitations in translating new findings on ADAMTS18 to clinical practice are also discussed.

Vitamin D and the Central Nervous System: Causative and Preventative Mechanisms in Brain Disorders

Nutrients

2022 Oct 17

Cui, X;Eyles, DW;
PMID: 36297037 | DOI: 10.3390/nu14204353

Twenty of the last one hundred years of vitamin D research have involved investigations of the brain as a target organ for this hormone. Our group was one of the first to investigate brain outcomes resulting from primarily restricting dietary vitamin D during brain development. With the advent of new molecular and neurochemical techniques in neuroscience, there has been increasing interest in the potential neuroprotective actions of vitamin D in response to a variety of adverse exposures and how this hormone could affect brain development and function. Rather than provide an exhaustive summary of this data and a listing of neurological or psychiatric conditions that vitamin D deficiency has been associated with, here, we provide an update on the actions of this vitamin in the brain and cellular processes vitamin D may be targeting in psychiatry and neurology.

Dicer ablation in Kiss1 neurons impairs puberty and fertility preferentially in female mice

Nature communications

2022 Aug 09

Roa, J;Ruiz-Cruz, M;Ruiz-Pino, F;Onieva, R;Vazquez, MJ;Sanchez-Tapia, MJ;Ruiz-Rodriguez, JM;Sobrino, V;Barroso, A;Heras, V;Velasco, I;Perdices-Lopez, C;Ohlsson, C;Avendaño, MS;Prevot, V;Poutanen, M;Pinilla, L;Gaytan, F;Tena-Sempere, M;
PMID: 35945211 | DOI: 10.1038/s41467-022-32347-4

Kiss1 neurons, producing kisspeptins, are essential for puberty and fertility, but their molecular regulatory mechanisms remain unfolded. Here, we report that congenital ablation of the microRNA-synthesizing enzyme, Dicer, in Kiss1 cells, causes late-onset hypogonadotropic hypogonadism in both sexes, but is compatible with pubertal initiation and preserved Kiss1 neuronal populations at the infantile/juvenile period. Yet, failure to complete puberty and attain fertility is observed only in females. Kiss1-specific ablation of Dicer evokes disparate changes of Kiss1-cell numbers and Kiss1/kisspeptin expression between hypothalamic subpopulations during the pubertal-transition, with a predominant decline in arcuate-nucleus Kiss1 levels, linked to enhanced expression of its repressors, Mkrn3, Cbx7 and Eap1. Our data unveil that miRNA-biosynthesis in Kiss1 neurons is essential for pubertal completion and fertility, especially in females, but dispensable for initial reproductive maturation and neuronal survival in both sexes. Our results disclose a predominant miRNA-mediated inhibitory program of repressive signals that is key for precise regulation of Kiss1 expression and, thereby, reproductive function.

Wholemount in situ Hybridization for Spatial-temporal Visualization of Gene Expression in Early Post-implantation Mouse Embryos

BIO-PROTOCOL

2021 Nov 18

Yang, X;Chen, Y;Song, L;Zhang, T;Jing, N;
| DOI: 10.21769/bioprotoc.4229

Wholemount _in situ_ hybridization has been widely used to explore gene expression distribution in both tissues and sections (Hauptmann and Gerster, 1994; Nieto _et al._, 1996). In the field of developmental biology, information on the spatial and temporal distribution of gene expression revealed by _in situ_ hybridization has facilitated the identification of master regulators of embryogenesis. In our recent study, we reported that _Pou3f1_ is an important regulator of mouse neuroectoderm development by combining wholemount _in situ_ hybridization and multiple functional analyses (Zhu _et al._, 2014). We optimized a wholemount RNA _in situ_ hybridization protocol that uses digoxigenin labeled RNA probes and an anti-digoxigenin antibody conjugated with alkaline phosphatase to detect the enrichment of _Pou3f1_ in the anterior embryonic region of the mouse gastrula, which indicated potential biological functions of _Pou3f1_ in embryonic ectoderm development. Thereafter, more lineage regulators of the mouse gastrulation have been revealed and validated using this optimized protocol (Yang _et al._, 2018 and 2019; Peng _et al._, 2016 and 2019). The current protocol exhibits strong experimental robustness and displays application potential in a wide range of biological studies. Thus, we summarize the protocol here, in the hope its application can facilitate the study of gene expression.

VEGF-B ablation in pancreatic?-cells upregulates insulin expression without affecting glucose homeostasis or islet lipid uptake

Sci Rep

2020 Jan 22

Ning FC1, Jensen N1, Mi J1, Lindstr�m W1, Balan M1, Muhl L1, Eriksson U1, Nilsson I1, Nyqvist D2.
PMID: 31969592 | DOI: 10.1038/s41598-020-57599-2

Type 2 diabetes mellitus (T2DM) affects millions of people and is linked with obesity and lipid accumulation in peripheral tissues. Increased lipid handling and lipotoxicity in insulin producing ?-cells may contribute to ?-cell dysfunction in T2DM. The vascular endothelial growth factor (VEGF)-B regulates uptake and transcytosis of long-chain fatty acids over the endothelium to tissues such as heart and skeletal muscle. Systemic inhibition of VEGF-B signaling prevents tissue lipid accumulation, improves insulin sensitivity and glucose tolerance, as well as reduces pancreatic islet triglyceride content, under T2DM conditions. To date, the role of local VEGF-B signaling in pancreatic islet physiology and in the regulation of fatty acid trans-endothelial transport in pancreatic islet is unknown. To address these questions, we have generated a mouse strain where VEGF-B is selectively depleted in ?-cells, and assessed glucose homeostasis, ?-cell function and islet lipid content under both normal and high-fat diet feeding conditions. We found that Vegfb was ubiquitously expressed throughout the pancreas, and that ?-cell Vegfb deletion resulted in increased insulin gene expression. However, glucose homeostasis and islet lipid uptake remained unaffected by ?-cell VEGF-B deficiency

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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