Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development

Abstract

Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development–related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a , which are required for bone development, and Rln1 and Irx5 , which had not been previously considered. Whereas Rln1 −/− mice exhibited small skeletal size only at birth, Irx5 −/− mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice.

Osteocyte Transcriptome Mapping Identifies a Molecular Landscape Controlling Skeletal Homeostasis and Susceptibility to Skeletal Disease

“…Analysis of the osteocyte transcriptome signature showed it was enriched with genes associated with skeletal biological processes in the GO database 40 (4.5 fold-enrichment (FE), P=1.0×10 -67 ), and with skeletal phenotypes in the Mouse Genome Informatics database 41 (MGI, 2.7 FE, P=4.7×10 -35 ). This included Sost , Dkk1 , Mepe and Dmp1 , genes known to be highly expressed in osteocytes, and genes with an established role in the skeleton, such as osteoprotegerin ( Tnfsf11b ), Wingless-type family member-1 ( Wnt1 ) 42 , fibroblast growth factor-9 ( Fgf9 ) 43 and Iroquois homeobox protein 5 ( Irx5 ) 44 ( Fig. 4a, Supplementary Fig 6 ).…”

Dysregulated Gene Expression of Imprinted and X-Linked Genes: A Link to Poor Development of Bovine Haploid Androgenetic Embryos

“…Uniparental haploid embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genome to early embryonic development. Moreover, haploid embryos have been used to derive embryonic stem cells and hold great promise for functional genetic studies and animal biotechnology ( Panneerdoss et al, 2012 ; Kokubu and Takeda, 2014 ; Bai et al, 2016 , 2019 ).…”

Irx3 and Irx5 - Novel Regulatory Factors of Postnatal Hypothalamic Neurogenesis

“…Due to the requirement of Irx3 and Irx5 in the development of many organs, including heart ( Costantini et al, 2005 ; Zhang et al, 2011 ; Gaborit et al, 2012 ; Kim et al, 2016 ) and bone ( Cain et al, 2016 ; Bai et al, 2019 ; Tan et al, 2020 ) which are also important for energy homeostasis, whole body knockout mice of Irx3 and Irx5 are both runty and thus not ideal for studying metabolic regulation. To circumvent this, a mouse line harboring cis -heterozygous mutant allele of Irx3 and Irx5 ( Irx3 + Irx5 + / Irx3 Δ Irx5 eGFP ; Irx3/5 dHet ) was employed to study the effects of half-reduction of the expression of Irx3 and Irx5 on energy homeostasis ( Son et al, 2021a ).…”