Embryonic Day Research Articles

Developmental mouse brain common coordinate framework

3D brain atlases are key resources to understand the brain’s spatial organization and promote interoperability across different studies. However, unlike the adult mouse brain, the lack of developing mouse brain 3D reference atlases hinders advancements in understanding brain development. Here, we present a 3D developmental common coordinate framework (DevCCF) spanning embryonic day (E)11.5, E13.5, E15.5, E18.5, and postnatal day (P)4, P14, and P56, featuring undistorted morphologically averaged atlas templates created from magnetic resonance imaging and co-registered high-resolution light sheet fluorescence microscopy templates. The DevCCF with 3D anatomical segmentations can be downloaded or explored via an interactive 3D web-visualizer. As a use case, we utilize the DevCCF to unveil GABAergic neuron emergence in embryonic brains. Moreover, we map the Allen CCFv3 and spatial transcriptome cell-type data to our stereotaxic P56 atlas. In summary, the DevCCF is an openly accessible resource for multi-study data integration to advance our understanding of brain development.

Spatiotemporal characteristics of eustachian tube development in C57BL/6 mice: Correlation between morphological and functional maturation.

The eustachian tube (ET), a critical conduit connecting the middle ear and nasopharynx, is essential for normal middle ear function. However, it remains one of the least understood anatomical structures due to its complexity and the challenges of in vitro manipulation. Historically, these challenges have hindered research into the morphology and function development of the ET. This study elucidates the spatiotemporal relationship of ET morpho-functional maturation in mice, identifying key periods and factors that lay the theoretical foundation for exploring the molecular mechanisms of ET-related diseases. We comprehensively characterized the ET development in C57BL/6 mice from embryonic day (E) 12.5 to postnatal day (P) 30, focusing on the development of cilia, secretory cells, surrounding glands, and macrophages. Immunostaining identified the localization and secretion patterns of the mucins Muc5b and Muc5ac within the ET. Additionally, using improved ET function assessment tools, we evaluated the developmental features of ET mucociliary clearance and ventilation functions. In C57BL/6 mice, E16.5 marks a critical period for middle ear cavity and ET formation. Muc5b plays a foundational role during early stages, while Muc5ac enhances function in later stages. During P7-11, despite morphological maturity, ET function remains underdeveloped but continues to improve with growth.

Heterozygous variants in the teashirt zinc finger homeobox 3 (TSHZ3) gene in human congenital anomalies of the kidney and urinary tract.

Around 180 genes have been associated with congenital anomalies of the kidney and urinary tract (CAKUT) in mice, and represent promising novel candidate genes for human CAKUT. In whole-exome sequencing data of two siblings with genetically unresolved multicystic dysplastic kidneys (MCDK), prioritizing variants in murine CAKUT-associated genes yielded a rare variant in the teashirt zinc finger homeobox 3 (TSHZ3) gene. Therefore, the role of TSHZ3 in human CAKUT was assessed. Twelve CAKUT patients from 9/301 (3%) families carried five different rare heterozygous TSHZ3 missense variants predicted to be deleterious. CAKUT patients with versus without TSHZ3 variants were more likely to present with hydronephrosis, hydroureter, ureteropelvic junction obstruction, MCDK, and with genital anomalies, developmental delay, overlapping with the previously described phenotypes in Tshz3-mutant mice and patients with heterozygous 19q12-q13.11 deletions encompassing the TSHZ3 locus. Comparable with Tshz3-mutant mice, the smooth muscle layer was disorganized in the renal pelvis and thinner in the proximal ureter of the nephrectomy specimen of a TSHZ3 variant carrier compared to controls. TSHZ3 was expressed in the human fetal kidney, and strongly at embryonic day 11.5-14.5 in mesenchymal compartments of the murine ureter, kidney, and bladder. TSHZ3 variants in a 5' region were more frequent in CAKUT patients than in gnomAD samples (p < 0.001). Mutant TSHZ3 harboring N-terminal variants showed significantly altered SOX9 and/or myocardin binding, possibly adversely affecting smooth muscle differentiation. Our results provide evidence that heterozygous TSHZ3 variants are associated with human CAKUT, particularly MCDK, hydronephrosis, and hydroureter, and, inconsistently, with specific extrarenal features, including genital anomalies.

Olig2+/NG2+/BLBP+ astrocyte progenitors: a novel component of the neurovascular unit in the developing mouse hippocampus.

Astrocytes are key components of the neurovascular unit. While we have recently identified Olig2+ astrocyte progenitors (ASPs) in the developing mouse dentate gyrus (DG), their molecular signature remains incompletely characterized. Here we demonstrate that Olig2+ ASPs predominantly express brain lipid-binding protein (BLBP), while only a small population of them expresses gfap-GFP. These Olig2+/BLBP+ ASPs co-express the transcription factors Sox3, Sox9 and the proteoglycan NG2 but not Sox10, a marker for oligodendrocyte progenitors (OLPs). Olig2+ ASPs appear from embryonic day 18 (E18) onwards and decline at postnatal day 14 (P14). Consistent with the proliferation of both Olig2+ and NG2+ glial cells after brain injury, intrauterine intermittent hypoxia (IH) led to an increase in Olig2+/NG2+/BLBP+ ASPs in the postnatal DG. IH also promoted both angiogenesis and vascular coupling of Olig2+/NG2+ ASPs. Our data suggest that IH-induced expression of HIF1a increases Olig2+/NG2+/BLBP+ ASPs in a cell non-autonomous manner. Our data also revealed increased vascular coupling of GFAP+ astrocytes following IH, while the number of GFAP+ astrocytes remains unchanged. Given that BLBP, Olig2 and NG2 are expressed in reactive astrocytes, our findings suggest that Olig2+/NG2+/BLBP+ ASPs represent a subtype of reactive astrocyte progenitors. Furthermore, the enhanced vascular coupling of Olig2+/NG2+/BLBP+ ASPs appears to be an adaptive response to hypoxic brain injury. This study provides new insights into the molecular characteristics of Olig2+/NG2+/BLBP+ ASPs and their potential role in the brain's response to hypoxic injury, contributing to our understanding of neurovascular unit dynamics in both development and pathological conditions.

Novel human PDX1 Asn196Thr variant causes pancreas agenesis and reduces the generation of duodenal enteroendocrine cells

Abstract Introduction/Objective Pancreatic and duodenal homeobox 1 (PDX1) is a well-studied transcription factor required for pancreas organogenesis. PDX1 mutations are associated with the development of diabetes. An 8-month-old female patient with previously uncharacterized compound heterogeneous PDX1 missense mutations (Thr151Met and Asn196Thr) was identified in this study. The patient has permanent neonatal diabetes, pancreas hypoplasia, and a malformed gallbladder. Methods/Case Report PDX1 Thr151Met and Asn196Thr variants in the patient were identified by next-generation sequencing gene panel and confirmed by Sanger sequencing. To examine how the mutations affect PDX1 functions in vitro, we expressed mouse Pdx1 Thr152Met and Asn197Thr, homologous to human PDX1 Thr151Met and Asn196Thr, respectively, in mouse insulinoma MIN6 cells and human embryonic kidney 293T cells and examined protein localization, protein stability, DNA binding, and protein-protein interaction. We also generated embryonic day 18 mouse embryos carrying Pdx1 Asn197Thr missense mutation using CRISPR/Cas9 to examine its roles in the development of the pancreas and other digestive organs in vivo. Results (if a Case Study enter NA) We found that the Pdx1 Asn197Thr variant, but not Thr152Met, altered its nuclear localization and disrupted PDX1-Onecut 1 interaction. Neither variant significantly affected PDX1 protein stability, but both reduced PDX1 binding to the Pdx1 gene promoter. Importantly, we found that the Pdx1 Asn197Thr variant caused pancreas agenesis and a reduction in the generation of enteroendocrine cells in the proximal duodenum in mouse models. Conclusion Our data indicated that the PDX1 Asn196Thr variant impairs its DNA binding and protein-protein interaction, causing pancreas agenesis and a reduction in the generation of duodenal enteroendocrine cells.

Jamming of nephron-forming niches in the developing mouse kidney creates cyclical mechanical stresses.

Urinary collecting tubules form during kidney embryogenesis through the branching of the ureteric bud epithelium. A travelling mesenchyme niche of nephron progenitor cells caps each branching ureteric bud tip. These 'tip domain' niches pack more closely over developmental time and their number relates to nephron endowment at birth. Yet, how the crowded tissue environment impacts niche number and cell decision-making remains unclear. Here, through experiments and mathematical modelling, we show that niche packing conforms to physical limitations imposed by kidney curvature. We relate packing geometries to rigidity theory to predict a stiffening transition starting at embryonic day 15 in the mouse, validated by micromechanical analysis. Using a method to estimate tip domain 'ages' relative to their most recent branch events, we find that new niches overcome mechanical resistance as they branch and displace neighbours. This creates rhythmic mechanical stress in the niche. These findings expand our understanding of kidney development and inform engineering strategies for synthetic regenerative tissues.

8490 The mRNA for the Nonsyndromic Deafness Gene Atp2b2 Is Reduced Following Developmental Hypothyroidism

Abstract Disclosure: E.A. Gregersen: None. R.P. Peeters: None. D. Forrest: None. D.S. Sharlin: None. Thyroid hormone (TH) is essential for auditory system development. In rodent models, low levels of TH during the early postnatal period delay cochlear tissue remodeling and alter ion channel function required for normal auditory function. TH receptors are ligand-dependent transcription factors that regulate gene expression. Considering this, auditory deficits observed in hypothyroidism are thought to be largely the result of altered expression of specific TH target genes. However, few TH-regulated genes in the developing cochlea have been reported. TH is reported to impact calcium signaling in several tissues, but whether regulators of calcium signaling are altered following developmental hypothyroidism in the cochlea is largely unexplored. In this report, we investigated the spatial and temporal expression of the ATPase, Ca++ transporting, plasma membrane 2 (Atp2b2) gene in a mouse model of developmental hypothyroidism. Timed-pregnant dams were treated with thyroid gland inhibitors to induce developmental TH insufficiency from embryonic day 12.5 (E12.5) through post-natal day 15 (P15). Untreated timed-pregnant dams served as euthyroid controls. Control and experimental cochleae were collected over a developmental period ranging from embryonic day 18.5 (E18.5) through P15. Cochleae were then processed to detect Atp2b2 mRNA by in situ hybridization or quantitative real-time PCR. In situ hybridization showed that Atp2b2 localizes to inner and outer hair cells as expected, but Atp2b2 was also observed at lower levels in the greater epithelial ridge and spiral ganglion. Quantification of Atp2b2 mRNA revealed an effect of postnatal day and treatment on Atp2b2. In euthyroid animals, Atp2b2 expression gradually increased until P10, then decreased slightly by P15. In hypothyroid animals, Atp2b2 mRNA was consistently decreased (2-2.5 fold). Hypothyroidism similarly impacted expression regardless of sex. The reduction in Atp2b2 mRNA expression in the hypothyroid animals suggests that Atp2b2 is developmentally regulated in part by thyroid hormone signaling. This study characterizes the relationship between TH and Atp2b2 expression during early cochlear development while providing insight into abnormal cochlear development due to hypothyroidism. Presentation: 6/3/2024

An atlas of small non-coding RNAs in human preimplantation development

Understanding the molecular circuitries that govern early embryogenesis is important, yet our knowledge of these in human preimplantation development remains limited. Small non-coding RNAs (sncRNAs) can regulate gene expression and thus impact blastocyst formation, however, the expression of specific biotypes and their dynamics during preimplantation development remains unknown. Here we identify the abundance of and kinetics of piRNA, rRNA, snoRNA, tRNA, and miRNA from embryonic day (E)3-7 and isolate specific miRNAs and snoRNAs of particular importance in blastocyst formation and pluripotency. These sncRNAs correspond to specific genomic hotspots: an enrichment of the chromosome 19 miRNA cluster (C19MC) in the trophectoderm (TE), and the chromosome 14 miRNA cluster (C14MC) and MEG8-related snoRNAs in the inner cell mass (ICM), which may serve as ‘master regulators’ of potency and lineage. Additionally, we observe a developmental transition with 21 isomiRs and in tRNA fragment (tRF) codon usage and identify two novel miRNAs. Our analysis provides a comprehensive measure of sncRNA biotypes and their corresponding dynamics throughout human preimplantation development, providing an extensive resource. Better understanding the sncRNA regulatory programmes in human embryogenesis will inform strategies to improve embryo development and outcomes of assisted reproductive technologies. We anticipate broad usage of our data as a resource for studies aimed at understanding embryogenesis, optimising stem cell-based models, assisted reproductive technology, and stem cell biology.

8235 Mutations in EMX2, a Homeodomain Transcription Factor, Cause Idiopathic Hypogonadotropic Hypogonadism

Abstract Disclosure: M. Stamou: None. M. Tompkin: None. H. Bow: None. H. Brand: None. L. Plummer: None. M. Talkowski: None. Y. Shen: None. D. Wu: None. R. Balasubramanian: None. S. Wray: None. S.B. Seminara: None. The genetic etiology of infertility remains largely unknown. To identify novel human infertility genes, we applied a de novo rare variant analysis in 142 parent-proband trios with idiopathic hypogonadotropic hypogonadism (IHH), a Mendelian disease caused by Gonadotropin Releasing Hormone (GnRH) deficiency. The discovery pipeline identified 43 rare de novo copy number variants (CNVs-deletions) spanning 263 genes and 213 rare de novo single nucleotide variants (SNVs) in 191 genes. Four genes were found to be affected by both rare de novo CNVs and SNVs among unrelated sporadic IHH probands. A rare variant association testing for protein truncating variants (PTVs) in the IHH cohort (N=1,394) compared to gnomAD controls (N=125,784) revealed that 3/4 genes demonstrated enrichment for PTVs in the IHH cohort compared to controls: the 2 known for IHH genes (ANOS1 and FGFR1) and one novel candidate gene, EMX2. IHH probands with de novo EMX2 variants demonstrated developmental delay and hearing loss. Common EMX2 variants were linked to delayed puberty/infertility, hearing loss and Parkinson’s disease in a phenome-wide association study of the Massachusetts General Brigham Biobank (N=65,253). Emx2 lineage tracing was conducted on Emx2Cre/+; RosatdT/+ and Emx2Cre/-; RosatdT/+ mouse embryos at embryonic day(E) 13.5. The tdT reporter was co-expressed in neuroendocrine GnRH (nGnRH) cells as they migrated from the olfactory pit to the nasal forebrain junction. WT and KO embryos from Emx2+/- matings were immunostained for GnRH across E12.5, E13.5, E14.5 and E16. The total number of nGnRH cells was greater in the Emx2 KO mice compared to WT littermates. However, by E16.5, fewer nGnRH cells were detected in the forebrain of Emx2 KO, compared to WT (WT= 496±28, KO=384±147, p=0.0125). Thus, failure of nGnRH neurons to enter the forebrain could contribute to the IHH pathogenesis. In conclusion, by utilizing a de novo variant analysis and a mouse model, EMX2 was uncovered as a novel gene for human infertility. Presentation: 6/1/2024

12547 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

8235 Mutations in EMX2, a Homeodomain Transcription Factor, Cause Idiopathic Hypogonadotropic Hypogonadism

Abstract Disclosure: M. Stamou: None. M. Tompkin: None. H. Bow: None. H. Brand: None. L. Plummer: None. M. Talkowski: None. Y. Shen: None. D. Wu: None. R. Balasubramanian: None. S. Wray: None. S.B. Seminara: None. The genetic etiology of infertility remains largely unknown. To identify novel human infertility genes, we applied a de novo rare variant analysis in 142 parent-proband trios with idiopathic hypogonadotropic hypogonadism (IHH), a Mendelian disease caused by Gonadotropin Releasing Hormone (GnRH) deficiency. The discovery pipeline identified 43 rare de novo copy number variants (CNVs-deletions) spanning 263 genes and 213 rare de novo single nucleotide variants (SNVs) in 191 genes. Four genes were found to be affected by both rare de novo CNVs and SNVs among unrelated sporadic IHH probands. A rare variant association testing for protein truncating variants (PTVs) in the IHH cohort (N=1,394) compared to gnomAD controls (N=125,784) revealed that 3/4 genes demonstrated enrichment for PTVs in the IHH cohort compared to controls: the 2 known for IHH genes (ANOS1 and FGFR1) and one novel candidate gene, EMX2. IHH probands with de novo EMX2 variants demonstrated developmental delay and hearing loss. Common EMX2 variants were linked to delayed puberty/infertility, hearing loss and Parkinson’s disease in a phenome-wide association study of the Massachusetts General Brigham Biobank (N=65,253). Emx2 lineage tracing was conducted on Emx2Cre/+; RosatdT/+ and Emx2Cre/-; RosatdT/+ mouse embryos at embryonic day(E) 13.5. The tdT reporter was co-expressed in neuroendocrine GnRH (nGnRH) cells as they migrated from the olfactory pit to the nasal forebrain junction. WT and KO embryos from Emx2+/- matings were immunostained for GnRH across E12.5, E13.5, E14.5 and E16. The total number of nGnRH cells was greater in the Emx2 KO mice compared to WT littermates. However, by E16.5, fewer nGnRH cells were detected in the forebrain of Emx2 KO, compared to WT (WT= 496±28, KO=384±147, p=0.0125). Thus, failure of nGnRH neurons to enter the forebrain could contribute to the IHH pathogenesis. In conclusion, by utilizing a de novo variant analysis and a mouse model, EMX2 was uncovered as a novel gene for human infertility. Presentation: 6/1/2024

7561 Maternal Sucrose Consumption During Gestation Alone Does Not Affect Glucocorticoids and Aldosterone in the Fetal Brain and Blood of Rats

Abstract Disclosure: M.M. Jung: None. D.R. Seib: None. M. Idrissi: None. H.W. Chen: None. K.K. Soma: None. Sucrose (table sugar) is a common added sugar in foods and beverages. Sucrose intake is high worldwide, yet little is known about how sucrose affects the brain and hormones. We designed an isocaloric, macro/micro-nutrient matched control diet (1% kcal sucrose) and high-sucrose diet (26% kcal sucrose) paradigm where the two diets only differ in kcal from sucrose. In this paradigm, maternal high-sucrose diet intake alters glucocorticoids (GCs) which are steroid hormones that regulate numerous physiological processes including the stress response. 16 wk of maternal sucrose intake (10 wk before mating, 3 wk during gestation, and 3 wk during lactation) increases blood and brain corticosterone levels in adult female offspring. Moreover, 13 wk of maternal sucrose intake (10 wk before mating and 3 wk during gestation) increases 11-deoxycorticosterone (DOC, corticosterone precursor) and 11-dehydrocorticosterone (DHC, corticosterone metabolite) in maternal serum, and increases aldosterone in fetal blood and brain at embryonic day 19.5 (E19.5). These results suggest that long-term maternal sucrose intake is a stressor that alters GC and aldosterone signalling. It is unclear whether a maternal high-sucrose diet only during gestation alters fetal steroid levels, or whether long-term sucrose consumption before mating is necessary. Here, we investigate the effects of maternal sucrose intake during gestation only on GCs and aldosterone in the fetal blood and brain. We hypothesize that maternal sucrose intake only during gestation will alter GC and aldosterone signaling in the fetus. We predict that maternal sucrose consumption during gestation alone will increase GC and aldosterone levels in the fetal blood and brain. Female Long-Evans rats were fed either a high-sucrose diet or a control diet starting E0.5 (day of mating confirmation) (n=15/diet). On E19.5, we collected fetal brain and blood and microdissected the regions of the fetal brain that are sensitive to GCs and aldosterone: nucleus accumbens, amygdala, hypothalamus, ventral hippocampus, and ventral tegmental area. We measured a panel of 15 steroids including GCs and aldosterone in the fetal brain (0.8-1.6 mg/region) and blood (5 µL) using liquid chromatography-tandem mass spectrometry, the gold standard for steroid quantification. Maternal high-sucrose diet intake during gestation alone did not alter maternal food intake, maternal body mass, or litter size, but increased the percentage of male offspring per litter. Maternal sucrose intake did not alter DOC, corticosterone, or DHC levels in the fetal blood and brain regions. Aldosterone was not detected in the fetal blood and brain. These results suggest that maternal sucrose intake during gestation alone does not alter GC and aldosterone levels in the developing fetus and that a longer duration of maternal sucrose intake is needed to alter offspring endocrine physiology. Presentation: 6/1/2024

12547 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

9264 The interplay of FOXO1 and NR3C1 in the development and function of pituitary somatotropes

Abstract Disclosure: P. Das: None. D.A. Jabal: None. D.A. Sunny: None. B.S. Ellsworth: None. Our research focuses on the role of glucocorticoid (GC) in regulating the maturation of pituitary somatotropes. We previously demonstrated that deletion of the forkhead transcription factor, FOXO1, affects the development of somatotropes both in vivo and in vitro. Our studies reveal that treating pregnant mice with the synthetic glucocorticoid, dexamethasone leads to premature somatotrope maturation. Interestingly, we have observed that the ability of GC to promote Gh1 expression is compromised in the absence of FOXO1 in a somatotrope-like cell line. This finding leads to further investigation into the GC receptor, NR3C1, and its developmental role in the pituitary gland. In mouse embryos, we detect NR3C1-positive cells localized primarily in the nucleus, initially in small numbers at embryonic day (e)12.5 and becoming more abundant by e14.5. At e14.5 there is a subset of cells where NR3C1 co-localizes with POU1F1, a marker of pre-somatotropes, suggesting the importance of NR3C1 in the formation of the somatotrope lineage. In dexamethasone-treated animals, we observe an increase in NR3C1-positive cells. However, no significant change was noted in POU1F1 expression. Significantly, the premature somatotropes identified are NR3C1 and POU1F1-positive, suggesting the pituitary cells expressing both POU1F1 and NR3C1 at e14.5 are likely differentiating into somatotropes when exposed to GC. Normally, by e16.5, GH-positive cells begin to appear in mice. Double immunostaining reveals that some of these cells also express NR3C1. Further supporting this finding, the deletion of Nr3c1 results in a decreased number of GH-positive cells at e16.5. Altogether these findings demonstrate that NR3C1 and FOXO1 are key factors regulating somatotrope maturation. Presentation: 6/2/2024

12674 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

6371 Conditional Deletion Of Nr5a1 In Sox9-cre Mice Impairs Adrenal Development

Abstract Disclosure: Y. Ikeda: None. A. Tagami: None. K. Ishizuka: None. M. Maekawa: None. The adrenal cortex is a major tissue that produces steroidogenic hormones necessary for regulating body homeostasis. Nuclear receptor subfamily 5, group A, member 1 (Nr5a1, SF-1, or Ad4BP) plays a crucial role in the regulation of adrenal and gonadal development, function and maintenance. SRY-related HMG-box gene 9 (Sox9) is upregulated by interaction of SRY with NR5A1, and leads the supporting cell precursors in XY gonads to differentiate into Sertoli-lineage cells at the initiation of testis differentiation. We previously generated Sox9-cre-Nr5a1flox/flox conditional knockout (cKO) mice, and showed that the loss of Nr5a1 in the gonads of XY Sox9-cre-Nr5a1flox/flox cKO mice abrogated the testicular pathway at embryonic day (E) 12.5–E13.5 to result in postnatal male-to-female gonadal sex reversal (1). Here, we investigated adrenal development of the Sox9-cre-Nr5a1flox/flox cKO mice. In mammals, after separation from the common adrenogonadal primordium, the adrenal primordium, which is formed dorsomedial to the gonadal primordium, begins to develop around E11.5. Double-immunostaining for NR5A1 and SOX9 revealed the presence of cells expressing both NR5A1 and SOX9 in the adrenal primordium of both sexes at E11.5 but not at E12.5. At E12.5, the number of NR5A1+ adrenocortical cells in Sox9-cre-Nr5a1flox/flox cKO mice markedly decreased compared with Nr5a1+/+ mice, and apoptotic cells, which were immunolabelled for active caspase 3, were detected in the area of the adrenal primordium in Sox9-cre-Nr5a1flox/flox cKO mice but not in Nr5a1+/+ mice, indicating that NR5A1- and SOX9-immunoreactive adrenocortical cells in Sox9-cre-Nr5a1flox/flox cKO mice were extinguished by apoptosis due to the activation of the Sox9-cre transgene. At E13.5–postnatal day 20, Sox9-cre-Nr5a1flox/flox cKO adrenals were hypoplastic associated with reduced number of NR5A1-immunoreactive adrenocortical cells, and only a few medullary cells immunolabelled for tyrosine hydroxylase, a marker of chromaffin cell precursors, were infiltrated into the adrenal primordium. Our findings suggest that sufficient number of adrenal cortical progenitors at the earliest stage of adrenal development are required for the subsequent adrenal development. In 8-week-old, Sox9-cre-Nr5a1flox/flox cKO adrenals were smaller in size compared with Nr5a1+/+ adrenals, but showed relatively normal organization with the medulla surrounded by the cortex. However, unlike XY Nr5a1+/+ adrenals, cells immunolabelled for the X-zone marker 20αHSD were observed at the junction between the cortex and medulla in XY Sox9-cre-Nr5a1flox/flox cKO adrenals, probably due to the sex reversal of the XY gonads, since androgen induces regression of the X-zone. Reference: (1) Ikeda et al., Sci Rep. 2021 Feb 24;11(1):4486. Presentation: 6/2/2024

12674 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&amp;S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024

5152 The interplay of FOXO1 and NR3C1 in the development and function of pituitary somatotropes

Abstract Disclosure: P. Das: None. D.A. Jabal: None. D.A. Sunny: None. B.S. Ellsworth: None. Our research focuses on the role of glucocorticoid (GC) in regulating the maturation of pituitary somatotropes. We previously demonstrated that deletion of the forkhead transcription factor, FOXO1, affects the development of somatotropes both in vivo and in vitro. Our studies reveal that treating pregnant mice with the synthetic glucocorticoid, dexamethasone leads to premature somatotrope maturation. Interestingly, we have observed that the ability of GC to promote Gh1 expression is compromised in the absence of FOXO1 in a somatotrope-like cell line. This finding leads to further investigation into the GC receptor, NR3C1, and its developmental role in the pituitary gland. In mouse embryos, we detect NR3C1-positive cells localized primarily in the nucleus, initially in small numbers at embryonic day (e)12.5 and becoming more abundant by e14.5. At e14.5 there is a subset of cells where NR3C1 co-localizes with POU1F1, a marker of pre-somatotropes, suggesting the importance of NR3C1 in the formation of the somatotrope lineage. In dexamethasone-treated animals, we observe an increase in NR3C1-positive cells. However, no significant change was noted in POU1F1 expression. Significantly, the premature somatotropes identified are NR3C1 and POU1F1-positive, suggesting the pituitary cells expressing both POU1F1 and NR3C1 at e14.5 are likely differentiating into somatotropes when exposed to GC. Normally, by e16.5, GH-positive cells begin to appear in mice. Double immunostaining reveals that some of these cells also express NR3C1. Further supporting this finding, the deletion of Nr3c1 results in a decreased number of GH-positive cells at e16.5. Altogether these findings demonstrate that NR3C1 and FOXO1 are key factors regulating somatotrope maturation. Presentation: 6/2/2024

Heterozygous variants in USP25 cause genetic generalized epilepsy.

USP25 encodes ubiquitin-specific protease 25, a key member of the deubiquitinating enzyme family that is involved in neural fate determination. Although abnormal expression in Down's syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown aetiology. Five heterozygous USP25 variants, including two de novo and three co-segregated variants, were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared with the East Asian population and all populations in the gnomAD database. The mean age at onset of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom, except that one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was expressed ubiquitously in mouse brain with two peaks, on embryonic Days 14-16 and postnatal Day 21, respectively. In human brain, likewise, USP25 is expressed in the fetus/early childhood stage and with a second peak at ∼12-20 years old, consistent with the seizure onset age in patients during infancy and in juveniles. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knockout mice, which showed increased seizure susceptibility compared with wild-type mice in a pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we used multiple functional detections. In HEK293 T cells, the variant associated with a severe phenotype (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed stable truncated dimers with an increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del variants increased neuronal excitability in mouse brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating that USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play an epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have a profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.

Mitochondrial DNA replication is essential for neurogenesis but not gliogenesis in fetal neural stem cells.

Mitochondria are unique organelles that have their own genome (mtDNA) and perform various pivotal functions within a cell. Recently, evidence has highlighted the role of mitochondria in the process of stem cell differentiation, including differentiation of neural stem cells (NSCs). Here we studied the importance of mtDNA function in the early differentiation process of NSCs in two cell culture models: the CGR8-NS cell line that was derived from embryonic stem cells by a lineage selection technique, and primary NSCs that were isolated from embryonic day 14 mouse fetal forebrain. We detected a dramatic increase in mtDNA content upon NSC differentiation to adapt their mtDNA levels to their differentiated state, which was not accompanied by changes in mitochondrial transcription factor A expression. As chemical mtDNA depletion by ethidium bromide failed to generate living ρ° cell lines from both NSC types, we used inhibition of mtDNA polymerase-γ by 2'-3'-dideoxycytidine to reduce mtDNA replication and subsequently cellular mtDNA content. Inhibition of mtDNA replication upon NSC differentiation reduced neurogenesis but not gliogenesis. The mtDNA depletion did not change energy production/consumption or cellular reactive oxygen species (ROS) content in the NSC model used. In conclusion, mtDNA replication is essential for neurogenesis but not gliogenesis in fetal NSCs through as yet unknown mechanisms, which, however, are largely independent of energy/ROS metabolism.