Activation of Proneuronal Transcription Factor Ascl1 in Maternal Liver Ensures A
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bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441617; this version posted April 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Activation of proneuronal transcription factor Ascl1 in maternal liver ensures a 2 healthy pregnancy 3 4 Joonyong Lee1, Veronica Garcia1, Shashank M. Nambiar1, Huaizhou Jiang1, and Guoli 5 Dai1 6 7 1Department of Biology, School of Science, Indiana University-Purdue University 8 Indianapolis, Indianapolis, IN 46202 9 10 Authors’ email 11 Joonyong Lee, PhD, Email: [email protected]; Veronica Garcia, Email: [email protected]; 12 Shashank M. Nambiar, Email: [email protected]; Huaizhou Jiang, PhD, Email: 13 [email protected]; and Guoli Dai, DVM, PhD, Email: [email protected]. 14 15 Corresponding author 16 Guoli Dai, DVM, PhD; 723 West Michigan Street, SL306. Indianapolis, IN 46202; Tel: 17 913-709-2906; Email: [email protected]. 18 19 Financial support 20 This work was supported by a grant from the National Institute of Diabetes and 21 Digestive and Kidney Diseases (1R01DK117076) and a pilot grant from the Center for 22 Diabetes and Metabolic Diseases of Indiana University School of Medicine. 23 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441617; this version posted April 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 25 Conflict of interest 26 No conflicts of interest, financial or otherwise, are declared by the authors. 27 28 Author contributions 29 Conceptualization: JL and GD. Investigation: JL, VG, SN, and HJ. Formal analysis: JL, 30 VG, and HJ. Data curation: JL, VG, and HJ. Writing: JL and GD. Resources: GD. 31 32 Abbreviations 33 Ascl1, achaete-scute homolog-like 1; IGF2, insulin-like growth factor 2; AST, aspartate 34 aminotransferase; ALT, alanine aminotransferase; PL, placental lactogen; GD, gestation 35 day. 36 37 Main text word count: 4,908 words including introduction, materials and methods, 38 results, and discussion. 39 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441617; this version posted April 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 40 41 Synopsis 42 43 How the maternal liver adapts to pregnancy remains elusive. We found that maternal 44 liver activates the expression of Ascl1, a gene encoding a proneuronal transcription 45 factor, to coordinate the adaptations of maternal organs and the growth of the placenta, 46 enabling a healthy pregnancy and normal postnatal growth of the offspring. 47 48 Abstract 49 50 Background & Aims: Maternal liver exhibits robust adaptations to pregnancy to 51 accommodate the metabolic needs of developing and growing placenta and fetus by 52 largely unknown mechanisms. We found that achaete-scute homolog-like 1 (Ascl1), a 53 basic helix-loop-helix transcription factor essential for neuronal development, is highly 54 activated in maternal hepatocytes during the second half of gestation in mice. Methods: 55 To investigate whether and how Ascl1 plays a pregnancy-dependent role, we deleted 56 the Ascl1 gene specifically in maternal hepatocytes from mid-gestation until term. 57 Results: As a result, we identified multiple Ascl1-dependent phenotypes. Maternal 58 livers lacking Ascl1 exhibited aberrant hepatocyte structure, increased hepatocyte 59 proliferation, enlarged hepatocyte size, reduced albumin production, and elevated 60 release of liver enzymes, indicating maternal liver dysfunction. Simultaneously, maternal 61 pancreas and spleen and the placenta displayed marked overgrowth; and the maternal 62 ceca microbiome showed alterations in relative abundance of several bacterial bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441617; this version posted April 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 63 subpopulations. Moreover, litters born from maternal hepatic Ascl1-deficient dams 64 experienced abnormal postnatal growth after weaning, implying an adverse pregnancy 65 outcome. Mechanistically, we found that maternal hepatocytes deficient for Ascl1 66 exhibited robust activation of insulin-like growth factor 2 expression, which may 67 contribute to the Ascl1-dependent phenotypes widespread in maternal and 68 uteroplacental compartments. Conclusion: In summary, we demonstrate that maternal 69 liver, via activating Ascl1 expression, modulates the adaptations of maternal organs and 70 the growth of the placenta to maintain a healthy pregnancy. Our studies reveal Ascl1 as 71 a novel and critical regulator of the physiology of pregnancy. 72 73 Keywords 74 Ascl1, hepatocyte, liver, pregnancy, insulin-like growth factor 2 75 76 77 78 79 80 81 82 83 84 85 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441617; this version posted April 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 86 87 Graphical Abstract 88 89 90 Introduction 91 92 The establishment and maintenance of pregnancy requires highly coordinated 93 adaptations in maternal, uteroplacental, and fetal compartments. As pregnancy 94 progresses, in the maternal compartment, the liver grows to expand its metabolic 95 capacity; the pancreas proliferates its β-cell population to elevate insulin production; the 96 gut alters its microbiome contributing to immunological adjustments; the spleen 97 undergoes development and growth of the erythroid lineage; and the kidney expands its 98 volume allowing for increases in blood flow and fluid retention 1-6. These adaptations of 99 maternal non-reproductive organs to pregnancy must be required to suffice the bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441617; this version posted April 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 100 demands of the developing and growing placenta and fetus. However, the physiological 101 significance and the regulatory mechanisms of these processes are poorly understood, 102 representing an emerging research field. 103 104 Our work and that of others have demonstrated that maternal liver undergoes 105 hyperplasia and hypertrophy, changes its gene expression profile, and thereby 106 markedly expands during gestation in rodents 7-9. However, how this is controlled 107 remains elusive. We previously showed that achaete-scute homolog-like 1 (Ascl1), a 108 basic helix-loop-helix transcription factor essential for neurogenesis 10-12, is highly 109 upregulated in maternal livers of pregnant rats 13. During development, Ascl1 controls 110 proliferation, cell cycle exit, and full neuronal differentiation and specification of neural 111 progenitor cells in both the central and peripheral nervous systems 11, 14, 15. Ascl1 112 inhibits its own expression by negative autoregulation in the developing nervous 113 system, possibly explaining the lack of overt abnormalities in Ascl1+/- mice 16, whereas 114 Ascl1-/- pups die within hours after birth due to defects in brain development 10. In the 115 adult, ASCL1 is expressed only in the brain and spinal cord where there is ongoing 116 neurogenesis, and in developing neuroendocrine cells in multiple organs including the 117 cerebellum, thyroid, and thymus 17. In this study, we evaluated the activation, and 118 explored the function, of Ascl1 in maternal livers of pregnant mice. We found that Ascl1 119 not only modulates maternal hepatic adaptation, but also mediates the communication 120 of maternal liver with other maternal organs and the placenta, eventually affecting 121 pregnancy outcomes. 122 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441617; this version posted April 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 123 124 Materials and methods 125 126 Mice 127 Ascl1fl/fl;R26EYFP/EYFP mice were a generous gift from Dr. Francois Guillemot (The 128 Francis Crick Institute, Midland Road, London NW 1AT, UK) 18. The mice are referred 129 hereafter as Ascl1fl/fl for simplicity. C57BL/6 mice were purchased from the Jackson 130 Laboratory (Bar Harbor, ME, USA). Mice were maintained on a 12-hour light/12-hour 131 dark cycle (7 AM on and 7 PM off) at 22 ± 1°C and given a standard rodent chow and 132 water ad libitum. All of the animal experiments were conducted in accordance with the 133 National Institutes of Health Guide for the Care and Use of Laboratory Animals. The 134 protocols for the care and use of animals were approved by the Indiana University- 135 Purdue University Indianapolis Animal Care and Use Committee. 136 137 Mouse genotyping 138 Genomic DNA was prepared from mouse ear snips using the modified HotSHOT 139 method 19. All mice were genotyped by polymerase chain reaction (PCR) using KAPA 140 Taq PCR Kits (Kapa Biosystems, Inc., Wilmington, MA, USA). Specific primers 141 purchased from Integrated DNA Technologies (Coralville, IA, USA) were used to detect 142 the wild type and mutant alleles (Supplemental Table 1). Primers Ascl1 Forward and 143 Ascl1 WT Reverse were used to detect the Ascl1 wild type allele (342 bp), and primers 144 Ascl1 Forward and Ascl1 Mutant Reverse were used to detect floxed Ascl1 allele (857 145 bp) 20. PCR conditions were 35 cycles of 94°C/30 sec; 69°C/30 sec; 72°C/90 sec. bioRxiv preprint doi: https://doi.org/10.1101/2021.04.27.441617; this version posted April 28, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.