Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence

Round 1

Reviewer 1 Report

The experimental work is well designed and the effect of Mcph1 Deficiency on Cell Cycle Arrest and Senescence via upregulation of pARF19 is demonstrated by robust integrated results. Minor criticisms regard formal aspects  such as: 1) scarce readability of a few sentences (the final tortuous sentence of the abstract-lines 28-32- should be split and reworded); 2) often incorrect nomenclature of the human (capital letters) and the mouse  (smaller letters) gene, use of "defect" instead of defective or deficients cells;  sentences clear in the meaning but faulty ( ex: line 45 that should be:  MCPH1  gene encoding  Micropephalin....  was the first reported to cause....); poor visibility of some Figures ( Fig.2C,G,H, Fig 3B            ). What do you mean for "homologous mutations" (line 47)?: replace homologous with "bi-allelic" and preferably use pathogenic variants instead of mutations. At line 77 bracr should be brca1. Line 69: replace MCPH1 with Microcephalin. Grammatically incorrect sentences should be reworded (Lines 112-114 ; 126-127). Either in the text or in the legend to Fig 3D indicate Satb2 (Special AT-Rich sequence-Binding protein 2). Line 214: multiple nuclei: do you mean micronuclei? Line 297: E2F1 "has" should be E2F1 is.......Lines 306-307 : what do you mean with only half of them were "unregulated"? all DEGs are unregulated: please explain. Line 249 : senesced?

The Authors are invited to revise the manuscript to increase  precision in delivering the key messages and readability.

Though not English speaking, I am sure that revision/editing by an English speaking person (colleague) can value  the scientific insights provided  by this work.

Author Response

Please see the attachment and the revised manuscript title"ijms-2935204 - Revision 1 ".

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript from Jiang et al examines Mcph1 knockout mice. The authors use existing Mcph1 knockout mice to examine the overall morphology and gene expression changes during embryonic development. This revealed changes to gene expression that could not be wholly accounted for by known interactions with the transcription factor E2F1. In addition, the authors create MEFs and find cell cycle deficits. Overall, the data in the manuscript are clear. However, there were several concerns noted, as detailed below.

Major:

My largest concern relates to the use of MEFS for a gene implicated in a neurodevelopmental disorder. A clearer justification for examining this cell type is needed. If the MCPH1 knockout pups are small during development, why examine postnatal MEFs?

What is the source of the “E2F1 target genes” dataset? Is this ChIP-seq data and from what cell type? Is there expression data that could also be included? Are the genes affected in MCPH1 knockout mice enriched for known E2F1 DNA binding sequence?

Are DEGs from the MCPH1 knockout RNA-seq data enriched for other targets of other transcription factors? ChIP-seq data may be available for (for example) components of the SWI/SNF complex.

In figure 3A, the intersection of DEGs and E2F1-bound genes is shown. Is this overlap statistically significant. A Fisher’s exact test should be used here.

On line 158, it is stated that genes related to neurodevelopment were chosen. How were these genes chosen? Are they involved in specific pathways related to neurodevelopment that would be related to the clinical features seen in patients? Genes implicated in “metabolic” pathways can impact cognition and were significantly enriched (Figure 3B) - why weren’t these examined?

Minor:

I suggest changing some terminology. For example, the word retardation, even when referring to growth can be offensive to some. Perhaps slowed or delayed growth? Similarly, “mental disability” should be altered to intellectual disability.

Mcph1 “defect” MEF cells should be altered for clarity. These are knockout cells, so should be described as such.

The text in dot blots figures 2 and 3 are too small. The top and bottom of panel 2G also seems to be cut off.

largely fine. There were a couple of phrases/words that I mentioned in my report. 

Author Response

Please see the attachment and the revised manuscript title"ijms-2935204 - Revision 1 "

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

authors have address my key major concerns