Dynamic changes in subplate and cortical plate microstructure at the onset of cortical folding in vivo

Reviewer #1 (Public review):

Summary:

This manuscript describes the analysis of fetal MRI and diffusion-weighted images of the fetal brain in utero, which reveals correlations between spatial and temporal patterns in diffusion behavior (associated with tissue microstructure) with local geometry of the brain surface (describing cortical folding). The authors use advanced imaging and image analysis pipelines, notably high angular resolution multi-shell diffusion imaging (HARDI) and multi-shell, multi-tissue constrained spherical deconvolution (MSMT-CSD) analysis of the resulting data to analyze. The key metric of tissue microstructure is "tissue fraction" which describes the relative contribution of organized anisotropic diffusion to overall diffusion, and the key geometry parameter is sulcal depth.

The major observation is that tissue fraction, which generally increases with gestational age, is lower in sulcal fundi, and importantly that the relative difference in tissue fraction emerges *before* folding occurs. The relatively low values of tissue fraction in regions of incipient sulci may be important to the physical mechanism of cortical folding.

Strengths:

Strengths of the manuscript include the application of advanced, highly technical imaging and image analysis methods to extract high-resolution data on both surface geometry and diffusion from a unique fetal cohort. The comparison of local features of surface and microstructure in both age-matched and age-mismatched analyses reveals a clear negative correlation between tissue fraction and sulcal depth.

Weaknesses:

The authors could improve the manuscript by (i) expanding their effort to place their current findings in the context of mechanistic models of folding and (ii) explaining more clearly how the diffusion measurements reflect tissue fraction. The relationship between the tissue fraction metric, the diffusion measurements, and the tissue microstructure is quite opaque.

Reviewer #2 (Public review):

Summary:

The authors analyze parameters related to anisotropy and gyrification in the developing human brain and describe an increase in tissue fraction (TF) across development. They correlate TF and sulcal depth in the CP and SP across local neighborhoods, describing a negative correlation. Also, they perform age-mismatched correlation of tissue fraction at early stages with sulcal depth at later ones and show correlation inside sulci, which they interpret as indicating the presence of minor structural changes in the brain that precede the development of sulci.

Strengths:

The study compiles a large cohort of cases through different developmental ages and performs sophisticated data analysis. Overall, the work is interesting.

Weaknesses:

I have some questions. What is the potential meaning of TF? It seems to be an estimator of anisotropy highly related to fractional anisotropy (FA), but it behaves in a complementary manner, increasing along gestation, in sharp contrast with the decrease observed in FA in this study (suppl. fig 3) and by others. Please clarify how it is calculated, what is the potential biological meaning of TF and how it differs from FA.

The correlations between TF and sulcal depth do not seem to provide much novelty, since as mentioned by the authors, previous evidence has pointed in that direction. The other concept in the paper relates to detecting structural changes in prospective sulcal areas in the cortex, which the authors analyze through the age-mismatched correlation of TF and subsequent sulcation. However, the results do not show a robust correlation as detailed below and do not seem particularly useful, as they require the inclusion of post-hoc information in the model, limiting the strength of the relationship and the predictive value. My main point of criticism is that if TF is a good marker of the structural modifications that will favor the development of sulci later in development, TF should show a map predictive of those sulci (e.g. at GW 25), that is however not the case. It is not necessary to correlate with future sulcal depth, as we know exactly where the primary sulci will develop. Conversely, it seems that TF decreases along the gyrification process, and it might just be a measure of the structural changes accompanying it.

In Figure 2 it illustrates the increase in TF across GA, but no R values or significance values are provided. Please add them to evaluate the robustness of the correlation.

In previous work of the authors, the subplate is not clearly distinguished from the subcortical white matter after 31 GW, as it starts to disintegrate (Kostovic et al., 2002; Calixto et al., 2024). However, in this manuscript, the SP is differentiated at those later ages. The methods section describes a 2 mm thick compartment below the cortical plate. However, if that is the case, it seems quite arbitrary (to coincide with the resolution of the diffusion imaging) and risks analyzing a compartment that is no longer present. Please explain the criteria followed for such distinction and more importantly, how such distinction is reliable considering the low detectability described in previous studies. In this regard, the discussion described that a rapid increase in TF was only seen in the SP after 30 GW, but maybe this increase would reflect the dissipation of the SP and the transformation of that space in subcortical white matter, with a much more expected anisotropy. The authors should review this.

The analysis describes a negative correlation between tissue fraction and sulcal depth when gyrification proceeds and the authors find that an age-mismatched correlation between tissue fraction in young embryos and sulcal depth in older embryos also shows a negative correlation in future sites of sulcation. However, for the correlation to exist, the tissue fraction in young individuals should already show low values in the prospective sulci, but no clear changes can be seen at GW 25 or 27 in lissencephalic areas that will bear sulci later on, as is the case of the central sulcus at GW 25 or the STS at GW 27, the latter showing very high tissue fraction (instead of the expected low).

Another question refers to Figures 3b and c. The graphs represent specific neighborhoods in the central sulcus at 27 and 35 GW. It can be argued that those neighborhoods might not be representative of the brain or of the whole sulcus. Please show the graph with all neighborhoods, which will provide more definitive evidence of the existence of the correlation. In this regard, the average graphs represented in Figure 3F seem to show a clear correlation at 27 GW in the subplate, but the correlation seems to fade at later stages (in both SP and CP), with both sulci and gyri exhibiting a negative correlation while other sulcal areas do not exhibit correlation. I think all points should be included in the correlation to better support the hypothesis.

Figure 4 shows the age-mismatched correlations, but they do not seem convincing particularly when they should be more useful, at 25 GW. Indeed, as seen in both Figures A and C, the central sulcus shows a negative correlation only in a few spots on one hemisphere, while (in C) most of the prospective sulcus shows a positive correlation, contrary to the hypothesis.

Lastly, the authors performed an age-mismatched correlation between TF at different ages and the sulcal depth at 35W, when it is maximal. This maximal depth might be "pushing" the correlation to significant territory. The authors should provide correlation also with the sulcal depth at other GAs, such as P29, P31, or P33, and analyze how the correlations hold.