Mcph1, Mutated in Primary Microcephaly, Is Required to Complete Terminal Erythroid Differentiation
Authors: Vial Yoann , Nardelli Jeannette , Rousselot Justine , Lepeu Emilie , Souyri Michele , Passemard Sandrine , Cave Helene , Drunat Severine 
Published: November 2019

Introduction: Microcephaly is a recurrent feature in patients with inherited bone marrow failure (iBMF) and DNA damage response (DDR) disorders suggesting that common cellular pathways regulate the proliferation and differentiation of neural and hematopoietic progenitors. However, while several studies addressed the role of iBFM or DDR genes in brain development, a possible role for microcephaly genes in hematopoietic development has not been investigated. To address this issue, we studied a mouse model of primary microcephaly with biallelic loss-of-function in Mcph1.MCPH1 mutations are found in 10% of patients with isolated forms of genetic microcephaly (MCPH). Interestingly, MCPH1 helps to maintain genomic integrity during cell division by interacting with proteins involved in cell cycle progression, apoptosis or DNA repair, all cellular processes being also involved in iBMF and DDR syndromes.
Methods: Mcph1 null mice were generated by germline deletion of Mcph1 exon 2 (Mcph1tm1.2Kali) (Liang et al., PLoS Genet., 2010). The subpopulations of erythroid progenitors S0 to S5 were phenotypically defined and sorted by flow cytometry according to CD71 and Ter119 expression in the Lin- compartment from mouse liver obtained at birth and during fetal development (E12.5). RNA sequencing (RNA-Seq) was performed on sorted erythroid progenitor fractions obtained from E12.5 fetal livers (SMARTer® Stranded Total RNA-Seq Kit V2-Pico Input library preparation kit). Cell division was studied by multiplexing erythroid specific antibodies with EdU flow cytometry cell proliferation assay.
Results: Null mice recapitulated the microcephaly phenotype seen in MCPH patients, but also showed a striking anemic pallor. Numeration and cytomorphologic examination of peripheral blood at birth confirmed macrocytic anemia with low red blood cell count and anisopoikilocytosis. These observations were consistent with congenital dyserythropoiesis in Mcph1-/- mice and prompted us to further characterize the erythroid lineage.
Quantification of erythroid progenitor populations in liver at birth showed a significant decreased from the S3 subset (Lin-, CD71High, Ter119High) suggesting impaired terminal differentiation. Similar results were obtained in fetal livers at E12.5 indicating that the defect arose early in hematopoietic ontogeny.
Transcriptome analysis of wild-type progenitor populations (S0 to S3) confirmed that Mcph1 is expressed during normal erythropoiesis following a Gata1-like expression profile. This is consistent with the presence in the Mcph1 gene promoter of a binding site for Gata1 and Ldb1 (ENCODE project), supporting an activation by the main erythroid differentiation complex. Strikingly, RNA-Seq analysis revealed deregulation of p53 pathway associated genes in all subsets of Mcph1-/- erythroid progenitors as compared to their wild-type counterparts. Two transcriptional p53 targets involved in cell cycle control, Cdkn1a coding the cyclin-dependent kinase inhibitor (p21) and Ccng1 coding Cyclin G1, were among the most upregulated genes.
Cell cycle analysis performed on sorted erythroid progenitors revealed an endoreduplication phenomenon restricted to the S3 subset with subsequent accumulation of tetraploid cells. Interestingly, physiological endoreduplication is initiated by p21 and E2Fs transcription factors, and Mcph1 functionally interacts with E2f1. Our findings suggest that, in the absence of Mcph1, Cdkn1a overexpression possibly combined to a decreased E2f1 activity may lead to endoreduplication in S3 progenitors, impairing further differentiation into mature red blood cells.
Few data are available for patients with MCPH1 mutations, hematological defects being possibly outlooked due to the severity of the neurological phenotype. However, CBC performed in one of our patients revealed a macrocytosis consistent with dyserythropoiesis evidenced in mice.
Conclusion: We demonstrate for the first time that Mcph1 expression is critical during terminal erythroid differentiation in mice. Altogether our findings provide additional evidence of a unique link between hematopoiesis and neuronal development.

Disclosures
No relevant conflicts of interest to declare.