Sibling chimerism among microglia in marmosets

Reviewer #1 (Public Review):

Summary:

Del Rosario et al characterized the extent and cell types of sibling chimerism in marmosets. To do so, they took advantage of the thousands of SNPs that are transcribed in single-nucleus RNA-seq (snRNA-seq) data to identify the sibling genotype of origin for all sequenced cells across 4 tissues (blood, liver, kidney, and brain) from many marmosets. They found that chimerism is prevalent and widespread across tissues in marmosets, which has previously been shown. However, their snRNA-seq approach allowed them to identify precisely which cells were of sibling origin, and which were not. In doing so they definitively show that sibling chimerism across tissues is limited to cells of myeloid and lymphoid lineages. The authors then focus on a large sample of microglia sequenced across many brain regions to quantify: (1) variation in chimerism across brain regions in the same individual, and (2) the relative importance of genetic vs. environmental context on microglia function/identity.

(1) Much like across different tissues in the same individual, they found that the proportion of chimeric microglia varies across brain regions collected from the same individuals (as well as differing from the proportion of sibling cells found in the blood of the same animals), suggesting that cells from different genetic backgrounds may differ in their recruitment and/or proliferation across regions and local tissue contexts, or that this may be linked to stochastic bottleneck effects during brain development.

(2) Their (admittedly smaller sample size) analyses of host-sibling gene expression showed that the local environment dominates genotype.

All told, this thoughtful and thorough manuscript accomplishes two important goals. First, it all but closes a previously open question on the extent and cell origins of sibling chimerism. Second, it sets the stage for using this unique model system to examine, in a natural context, how genetic variation in microglia may impact brain development, function, and disease.

The conclusions of this paper are well supported by the data, and the authors exert appropriate care when extrapolating their results that come from smaller samples. However, there are a few concerns that should be addressed.

The "modest correlation" mentioned in lines 170-172 does not take into account the uncertainty in estimates of each chimeric cell proportion (although the plot shows those estimates nicely). This is particularly important for the macrophages, which are far less abundant. Perhaps a more appropriate way to model this would be in a binomial framework (with a random effect for individuals of origin). Here, you could model the sibling identity of each macrophage as a function of the proportion of sibling-origin microglia and then directly estimate the percent variance explained.

A similar (albeit more complicated because of the number of regions being compared) approach could be applied to more rigorously quantify the variation in chimerism across brain regions (L198-215; Figure 4). This would also help to answer the question of whether specific brain regions are more "amenable" to microglia chimerism than others.

While the sample size is small, it would be exciting to see if any microglia eQTL are driven by sibling chimerism across the marmosets.

L290-292: The authors should propose ways in which they could test the two different explanations proposed in this paragraph. For instance, a simulation-based modeling approach could potentially differentiate more stochastic bottleneck effects from recruitment-like effects.

While intriguing, the gene expression comparison (Figure 5) is extremely underpowered. It would be helpful to clarify this and note the statistical thresholds used for identifying DEGs (the black points in the figure).

Reviewer #2 (Public Review):

Summary:

This manuscript reports a novel and quite important study of chimerism among common marmosets. As the authors discuss, it has been known for years that marmosets display chimerism across a number of tissues. However, as the authors also recognize, the scope and details of this chimerism have been controversial. Some prior publications have suggested that the chimerism only involves cells derived from hematopoietic stem cells, while other publications have suggested more cell types can also be chimeric, including a wide range of cell types present in multiple organs. The present authors address this question and several other important issues by using snRNA-seq to track the expression of host and sibling-derived mRNAs across multiple tissues and cell types. The results are clear and provide strong evidence that all chimeric cells are derived from hematopoietic cell lineages.

This work will have an impact on studies using marmosets to investigate various biological questions but will have the biggest impact on neuroscience and studies of cellular function within the brain. The demonstration that microglia and macrophages from different siblings from a single pregnancy, with different genomes expressing different transcriptomes, are commonly present within specific brain structures of a single individual opens a number of new opportunities to study microglia and macrophage function as well as interactions between microglia, macrophages, and other cell types.

Strengths:

The paper has a number of important strengths. This analysis employs the first unambiguous approach providing a clear answer to the question of whether sibling-derived chimeric cells arise only from hematopoietic lineages or from a wider array of embryonic sources. That is a long-standing open question and these snRNA-seq data seem to provide a clear answer, at least for the brain, liver, and kidney. In addition, the present authors investigate quantitative variation in chimeric cell proportions across several dimensions, comparing the proportion of chimeric cells across individual marmosets, across organs within an individual, and across brain regions within an individual. All these are significant questions, and the answers have important implications for multiple research areas. Marmosets are increasingly being used for a range of neuroscience studies, and a better understanding of the process that leads to the chimerism of microglia and macrophages in the marmoset brain is a valuable and timely contribution. But this work also has implications for other lines of study. Third, the snRNA-seq data will be made available through the Brain Initiative NeMO portal and the software used to quantify host vs. sibling cell proportions in different biosamples will be available through GitHub.

Weaknesses:

I find no major weaknesses, but several minor ones. First, the main text of the manuscript provides no information about the specific animals used in this study, other than sex. Some basic information about the sources of animals and their ages at the time of study would be useful within the main paper, even though more information will be available in the supplementary material. Second, it is not clear why only 14 pairs of animals were used for estimating the correlation of chimerism levels in microglia and macrophages. Is this lower than the total number of pairwise comparisons possible in order to avoid using non-independent samples? Some explanation would be helpful. Finally, I think more analysis of the consistency and variability of gene expression in microglia across different regions of the brain would be valuable. Are there genetic pathways expressed similarly in host and sibling microglia, regardless of region of the brain? Are there pathways that are consistently expressed differently in host vs sibling microglia regardless of brain region?