Author response: Spatiotemporal tissue maturation of thalamocortical pathways in the human fetal brain

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The development of connectivity between the thalamus and maturing cortex is a fundamental process in the second half of human gestation, establishing the neural circuits that are the basis for several important brain functions. In this study, we acquired high-resolution in utero diffusion magnetic resonance imaging (MRI) from 140 fetuses as part of the Developing Human Connectome Project, to examine the emergence of thalamocortical white matter over the second to third trimester. We delineate developing thalamocortical pathways and parcellate the fetal thalamus according to its cortical connectivity using diffusion tractography. We then quantify microstructural tissue components along the tracts in fetal compartments that are critical substrates for white matter maturation, such as the subplate and intermediate zone. We identify patterns of change in the diffusion metrics that reflect critical neurobiological transitions occurring in the second to third trimester, such as the disassembly of radial glial scaffolding and the lamination of the cortical plate. These maturational trajectories of MR signal in transient fetal compartments provide a normative reference to complement histological knowledge, facilitating future studies to establish how developmental disruptions in these regions contribute to pathophysiology. Editor's evaluation This study presents important new findings regarding prenatal thalamocortical development. The authors present convincing evidence, while overcoming substantial methodological challenges, in charting prenatal brain development in vivo. This work will be of interest to pediatric and developmental neuroscientists and neuroradiologists. https://doi.org/10.7554/eLife.83727.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Thalamocortical connections provide important inputs into the developing cortex during the second half of human gestation, where they play a key role in guiding cortical areal differentiation and establishing the circuitry responsible for sensory integration across the lifespan (Jones, 2007; Price et al., 2006; Schummers et al., 2005; Sharma et al., 2000; Sur and Rubenstein, 2005). Their importance is highlighted by previous work implicating disruptions to thalamocortical development during the perinatal period in the pathophysiology of neurodevelopmental disorders, such as schizophrenia (Klingner et al., 2014; Marenco et al., 2012), bipolar disorder (Anticevic et al., 2014), and autism (Nair et al., 2013). Altered thalamocortical connectivity has also been described in preterm infants, and was used to predict cognitive outcome (Ball et al., 2013; Ball et al., 2015; Toulmin et al., 2021), highlighting the specific vulnerability of these pathways during the second to third trimester. Although thalamocortical development has been studied in rodents and non-human primates (Brody et al., 1987; Kostović and Jovanov-Milosević, 2006; Molnár and Blakemore, 1995; Yakovlev et al., 1960) and post-mortem human tissue (Krsnik et al., 2017; Takahashi et al., 2012; Wilkinson et al., 2017), little is known about in vivo white matter maturation during fetal development. White matter development in the late second and third trimesters of human gestation (between 21 and 37 weeks) is characterised by a sequence of precisely timed biological processes occurring in transient compartments of the fetal brain. These processes include the migration of neurons along the radial glial scaffold, accumulation of thalamocortical axons in the superficial subplate, innervation of the target cortical area, conversion of radial glial cells into astrocytes, and ensheathment of axonal fibres (Krsnik et al., 2017; Molliver et al., 1973; Kostović et al., 2002, Kostovic and Judas, 2006). The challenge for in vivo neuroimaging studies is to disentangle the effect of these different neurobiological processes on the diffusion magnetic resonance imaging (dMRI) signal, to improve mechanistic insight about the transformation of transient fetal compartments into segments of developing white matter (Kostovic 2012). Recent advances in diffusion weighted imaging now allow in vivo characterisation and estimation of white matter development during the fetal period. Tractography has been used to estimate the fetal brain’s major white matter bundles and quantitatively characterise the evolution of the microstructure across the second half of gestation (Bui et al., 2006; Zanin et al., 2011; Jaimes et al., 2020; Jakab et al., 2015; Keunen et al., 2018; Khan et al., 2019; Machado-Rivas et al., 2021; Wilson et al., 2021). Advanced acquisition and analysis methods enable the relative contribution of constituent tissue and fluid compartments to the diffusion signal to be estimated (Jeurissen et al., 2014; Pietsch et al., 2019). Using this approach, previous work has identified non-linear trends in diffusion metrics over the second to third trimester (Wilson et al., 2021). Namely, we observed an initial decrease in tissue fraction within developing white matter between 22 and 29 weeks, which could be due to the radial glial scaffold disassembling (Rakic, 2003). Subsequently, we observed an increase from 30 to 36 weeks, potentially linked to more coherent fibre organisation, axonal outgrowth, and ensheathment (Wimberger et al., 1995; Back et al., 2002; Haynes et al., 2005), increasing the structural integrity of maturing white matter. Interpreting these trends is especially challenging in the rapidly developing fetal brain, because of the high sensitivity and low specificity of diffusion metrics to various co-occurring biological processes. We hypothesise that the biological processes occurring in different fetal compartments leads to predictable changes in diffusion metrics along tracts, reflecting the appearance and resolution of these transient zones. When a mean value across the whole tract is calculated, sensitivity to the unique neurobiological properties of each transient compartment is lost. For example, in the early prenatal and mid prenatal period, the subplate is a highly water-rich compartment containing extracellular matrix, whereas the cortical plate and the deep grey matter are relatively cell dense (Kostović, 2020). We therefore predict that the tissue fraction would be higher in the deep grey matter and the cortical plate and lower in the subplate. We investigate this by characterising the entire trajectory of tissue composition changes between the thalamus and the cortex, to explore the role of transient fetal brain developmental structures on white matter maturational trajectories. We acquired diffusion weighted imaging from 140 fetuses over a wide gestational age (GA) range (21–37 weeks) and use tractography to delineate five distinct thalamocortical pathways. To investigate whether the immature axonal bundles can be traced back to specific and distinct locations within thalamus, we parcellate the thalamus according to streamline connectivity (Behrens et al., 2003). We find consistent and distinct origins of different tracts, resembling the adult topology of thalamic nuclei (Toulmin et al., 2015; Behrens et al., 2003) as early as 23 weeks’ gestation. We then apply a multi-shell multi-tissue constrained spherical deconvolution (MSMT-CSD) diffusion model (Jeurissen et al., 2014) and derive tissue and fluid fraction values, charting tract-specific maturational profiles over the second to third trimester. We overlay the tracts on an atlas of transitioning fetal compartments and correlate changes in the dMRI signal across time with critical neurodevelopmental processes, such as the dissolution of the subplate and lamination of the cortical plate. We demonstrate that along-tract sampling of diffusion metrics can capture temporal and compartmental differences in the second to third trimester, reflecting the maturing neurobiology of the fetal brain described in histological studies. With these methods, we provide a detailed, accurate reference of the unique developing microstructure in each tract that improves mechanistic insight about fibre maturation, bridging the gap between MRI and histology. Results Estimating thalamocortical pathways using probabilistic streamline tractography High-angular-resolution multi-shell diffusion weighted imaging (HARDI) was acquired from 140 fetuses between 21 and 37 gestational weeks (70 male, 70 female) as part of the Developing Human Connectome Project (dHCP). Data were corrected for fetal head motion and other imaging artefacts (Christiaens et al., 2021). Individual subject orientation density functions (ODFs) were then computed using cohort-specific fluid and ‘tissue’ response functions and compiled to generate weekly diffusion templates (see Materials and methods). The diffusion templates were then registered to a T2-weighted brain atlas (Gholipour et al., 2017) of tissue segmentations, used to generate anatomically constrained whole-brain connectomes for each gestational week (Smith et al., 2012; Tournier et al., 2019). To constrain our investigation, we selected thalamocortical pathways that are at a critical stage in their development and are vulnerable to external influences in the second to third trimester (Batalle et al., 2017; Nosarti et al., 2014; Raybaud et al., 2013), the anterior thalamic radiation (AT), thalamic-motor tract (TM), thalamic-sensory tract (TS), posterior parietal tract (PP), and optic radiation (OR). The connectomes were filtered down to the pathways of interest using inclusion regions defined by the T2 atlas, including the thalamus and specific cortical areas (Figure 1). These included the primary motor cortex, primary sensory cortex, posterior parietal cortex, dorso-lateral prefrontal cortex, and the primary visual cortex. With this method, we were able to delineate five major thalamocortical pathways in each gestational week. To keep regions of interest more consistent across the cohort, we grouped all cases into 2-weekly intervals, starting at 23 weeks (Figure 2), replicating methods used previously (Wilson et al., 2021). Figure 1 Download asset Open asset Methods pipeline to estimate and quantify thalamocortical tracts development. (Top row) (a) Whole-brain connectomes generated for each gestational week template. (b) Atlas-defined masks of the thalamus and cortical areas were used to extract white matter pathways of interest from the connectomes. (c) These pathways were transformed to the native fetal diffusion space, (d) the values were sampled along the tract. (f) Whole-tract average diffusion metrics were calculated or (g) values sampled along the tract were aligned to an atlas of transient fetal compartments. Figure 2 Download asset Open asset Tractography of thalamocortical pathways in different gestational week templates across the second to third trimester. Tracts project to five different cortical areas, the prefrontal cortex, primary motor cortex, primary sensory cortex, posterior parietal cortex, and primary visual cortex, coloured according to the anterior-posterior axis. Structural connectivity parcellation of the fetal thalamus resembles adult topology of thalamic nuclei Tract density imaging (Calamante et al., 2010) was used in each ODF template to explore whether the different cortical areas were connected to distinct, specific regions of the thalamus (Figure 3a). We found that for all ages, there was symmetrical topographical representation of the cortical regions of interest in the thalamus. Furthermore, they spatially corresponded to the adult organisation of thalamic nuclei, demonstrated by the schematic (Figure 3a) which is based on Morel’s thalamus and other connectivity-derived parcellations from adult imaging studies (Morel et al., 1997; Najdenovska et al., 2018; Niemann et al., 2000). The tract projecting to the prefrontal cortex was connected to the anterior thalamus and in the younger ages (23–29 weeks) also to the medial thalamus. In the older templates (31, 33, and 35 weeks), frontal connectivity was more localised to the anterior thalamus and less evident in the medial area. There were distinct but neighbouring areas in the ventral thalamus connecting to the sensory and motor cortical areas, the motor-connected thalamic region being more frontal. The connectivity of the posterior parietal area was in the posterior part of the thalamus, and the most posterior voxels in the thalamic mask projected to the primary visual cortex. Figure 3 Download asset Open asset Tract density imaging parcellation of thalamus at different fetal ages. (a) A schematic of expected cortical connectivity arrangement across the thalamus, based on Morel’s parcellation of the adult thalamic nuclei. (b) Axial slices of thalamic parcellation, thresholded for the top 20% of voxels, colour-coded according to streamline connectivity of different tracts at 23 weeks, (c) 27 weeks, (d) 31 weeks, and (e) 35 weeks. Whole-tract average diffusion metrics have a characteristic U-shaped trend across the second to third trimester The thalamocortical pathways were transformed from the age-matched templates to the native subject space for 140 fetal subjects (Figure 4a). The MSMT-CSD-derived voxel-average tissue and fluid ODF values were sampled along the warped group-average streamline tracts. Tract-specific values were derived by averaging these for each tract in each subject, replicating the approach that has been used in previous fetal studies (Wilson et al., 2021). The values for each tract were plotted against the GA of the subject. The Akaike information criterion suggested second-order polynomial relationships for all tracts for both tissue and fluid fraction metrics, except the fluid fraction in the AT which is linear (Figure 4b). Diffusion tensor metrics also displayed similar age-related polynomial trends (Figure 4—figure supplement 1). We compared male vs. female (two-sample test) in each gestational week and found no significant differences (p>0.1). Figure 4 with 1 supplement see all Download asset Open asset Diffusion metric age trajectories for each tract. (a) Distribution of age among the fetal cohort (n=140) in gestational weeks. (b) Whole-tract average tissue (top) and fluid fractions (bottom) for each subject in the left (orange) and right (blue) hemisphere, plotted against gestational age (GA) of the subject, best fit by second-order polynomials (AT = anterior thalamic radiation, OR = optic radiation, PP = posterior parietal tract, TS = thalamic-sensory tract, TM = thalamic-motor tract). Along-tract sampling reveals evolving properties of fetal brain transient compartments To explore the origins of these trends in diffusion metrics, the values of tissue and fluid fraction were sampled in subject space at 100 equidistant intervals between the thalamus and the cortex. Tissue and fluid fraction are scaled jointly per scan such that they are approximately reciprocal of one another across the brain using a cubic polynomial spatial model (Pietsch et al., 2019). In each subject, we sampled the tissue and fluid fraction values beneath the streamlines from the thalamus to the cortex, plotting the microstructural tissue composition against the distance from the thalamus (Figure 5). We found that trajectories changed gradually between gestational weeks, and therefore we grouped them to match previous histological studies that define this fetal period according to three developmental windows, early (21–25.5 weeks), mid (26–31.5 weeks), and late (32–36 weeks) prenatal period (Kostović, 2020; Appendix 1—figures 5 and 6). When comparing the microstructural profiles of all the tracts in the different periods, the motor, sensory, and parietal tracts shared similar trajectories, whilst those in the AT and OR tracts were more distinct (Figure 5a, b, and c and Figure 5—figure supplement 1a,b). Figure 5 with 1 supplement see all Download asset Open asset Microstructural composition of fetal compartments traversed by developing thalamic white matter. Tracts were overlayed on the atlas of fetal compartments (examples highlight the difference between fetal brain structure in early prenatal [25 weeks] on far left, and late prenatal [35 weeks] on far right). Tissue fraction trends (top row) and fluid fraction trends (bottom row), normalised to 1, between the thalamus and cortex (thalamocortical tract axis) for the (a) thalamic-motor tract, (b) optic radiation, and (c) anterior thalamic radiation. Subjects were grouped by age, and average trajectories plotted for early prenatal (22–25.5 weeks), mid prenatal (26–31.5 weeks), late prenatal (32–36 weeks). Error bars represent the standard deviation among all subjects in each group. Atlas-derived tissue boundaries are marked on the trajectories to reveal the changing tissue properties of each layer between early, mid, and late prenatal development (cortical spinal fluid = CSF, cortical plate = CP, subplate = SP, intermediate zone = IZ, ventricular zone = VZ, deep grey matter = GM, immature white matter = WM). To improve our ability to corroborate changes in the dMRI signal with observations from histological studies, we mapped the maturational trajectories to an atlas of fetal brain compartments (Gholipour et al., 2017) and overlayed the boundaries of these compartments on the tissue and fluid fraction trajectories (Figure 5). By overlaying the tracts on the atlas, and corroborating each sampling segment with an atlas region, we could characterise the diffusion properties of the different fetal compartments independent of their change in size across gestation. Tissue fraction values in the deep grey matter and the cortical plate areas increased with GA in all tracts. This increase was most marked in the tracts terminating in superior areas of the brain (motor, sensory, and superior parietal cortex) (Figure 5a TM, Figure 5—figure supplement 1 (a) TS and (b) PP). The tissue fraction of the ventricular and intermediate zones decreased between the early and mid prenatal period, in all tracts. This decrease was very pronounced in the motor, sensory, and superior parietal tracts. The subplate tissue fraction changes were more tract specific. In the subplate of sensorimotor and parietal tracts, there was initially a very high fluid fraction and low tissue fraction, which transitions across the second to third trimester, increasing in tissue fraction from early to mid and then to late prenatal. Whereas in the AT, there was a decrease in subplate tissue fraction with GA (and a reciprocal increase in fluid fraction). In the OR, the subplate tissue fraction decreases between early and mid prenatal to then increase again in late prenatal. Highest tissue fractions were generally observed in the ventricular zone, with the lowest tissue fraction in the subplate area. To statistically test if there was a difference between the values in each compartment across GA, we correlated the tissue fraction values against age for each of the 100 sampling points. After correcting for multiple comparisons, points 1–16, 24–42, and 70–100 had significant linear correlations with age. Sampling points 43–69 had significant second-order polynomial relationships with age. Discussion In this work, we studied in utero development of five distinct thalamocortical pathways using state-of-the-art dMRI methods and bespoke pre-processing pipeline (Christiaens et al., 2019a; Cordero-Grande et al., 2019; Hutter et al., 2018a; Pietsch et al., 2019; Wilson et al., 2021) in 140 fetuses aged 21–37 weeks’ gestation. We show that these pathways connect to distinct thalamic nuclei, which could be clearly defined at group level even at 23 weeks. To disentangle the impact of different neurobiological processes on diffusion metrics, we characterised the tissue composition profile along each of the thalamocortical tracts as they traverse the different developmental tissue layers of the fetal brain. We used MSMT-CSD to model this fetal DWI dataset because it does not mandate a specific set of b-values. The technique exploits the unique b-value dependencies of different tissue types, and so depends inherently on the characteristics of the tissue in the fetal brain. The distinct properties of different fetal compartments after the application of MSMT-CSD are highlighted by the ODFs (Appendix 1—figure 4). Readers are directed to Tournier et al., 2019, for a more comprehensive review of the optimisation process involved to select appropriate b-shell values for the acquisition. We found that the spatiotemporal changes in the diffusion signal reflected known developmental processes that take place between the early, mid, and late prenatal period. The early period is characterised by higher tissue fractions in the middle of the tract, where there is a radial scaffold for migrating neurons. As this scaffold dissipates in the mid prenatal period, this is accompanied by a reduction in the tissue fraction in the middle of the tract, and an increase in tissue fraction towards the termination of the tracts as the neurons of the cortical plate mature. Finally in the late prenatal period, we observe the highest tissue fraction values at the start and end of the axis, as the pre-myelination phase of white matter development commences. This study demonstrates how the dMRI signal can be modelled to create in vivo spatiotemporal trajectories which relate to underlying neurobiological properties and are consistent with described trends from post-mortem histology (Kostović, 2020). Early embryonic patterning of gene expression and cell division in the thalamus provide a template for specialised nuclei to emerge over the course of development, such that specific cells eventually occupy distinct locations within the thalamus (Clascá et al., 2012; Nakagawa, 2019, Kostovic and Goldman-Rakic, 1983). Rodent studies labelling the embryonic thalamus demonstrated that there is a characteristic topography of thalamic projections which roughly exists at their time of arrival; anterior to posterior movement along the convexity of the cortex is represented in a medial-to-lateral axis within the thalamus, while ventral-dorsal movement across the cortex is represented in an anterior-to-posterior axis within the thalamus (Molnár et al., 1998; Molnár et al., 2012). Thalamocortical tracts emerge over the same timescale as the thalamus parcellates and matures into its specialised group of nuclei (Clascá et al., 2012). Although the topography of thalamic nuclei and their cortical connectivity is acquired embryonically, no in vivo parcellation of the thalamus in the fetal brain has been published. Using tract density imaging, we observed that the cortical areas were connected to specific thalamic regions, organised in an anterior-posterior axis. This anterior-posterior representation of cortical connectivity in the thalamus was consistent across the second to third trimester and is in accordance with the topology of thalamic nuclei described in rodent studies and histology (Molnár and Blakemore, 1995; Molnár et al., 1998). In addition, our fetal structural connectivity parcellation resembles the functionally derived thalamic parcellation in neonates, supporting the view that there is a strong association between structure and function in thalamocortical circuitry that begins early in life (Johansen-Berg et al., 2005; Toulmin et al., 2015; Alcauter et al., 2014). However, it is worth noting that this thalamic parcellation is dependent on streamline count through a voxel, and in the fetal brain streamlines are prone to spurious detection. This limitation is particularly relevant in the youngest fetuses, where we observe an extremely dense connectome (due to a fixed number of streamlines in a smaller brain) but there are very few coherent axonal bundles, so tracts might be overrepresented in the thalamic parcellation. The topography of thalamic nuclei is also not static during the embryonic and fetal period (Le Gros Clark, 1936), as pulvinar size increases and the dorsal lateral geniculate nucleus shifts its position from dorsolateral to ventromedial (Rakic, 1977; Mojsilović and Zecević, 1991). However, the image resolution of this study and the timespan in development over which this data was collected limit us from visualising these differences across age. Recent studies characterising developing white matter pathways using human fetal MRI identified second-order polynomial maturational trends in diffusion metrics unique to this developmental period (Wilson et al., 2021; Machado-Rivas et al., 2021). Here, we replicated these methods with a different group of tracts and found the same U-shaped trends in thalamocortical white matter development. The inflection point at around 29–30 weeks was hypothesised to be the result of the dissipating radial glial scaffold followed by the pre-myelination phase of white matter development (Wilson et al., 2021; Machado-Rivas et al., 2021). The sensitivity of HARDI to radially organised structure in the fetal brain has been described by previous studies (Miyazaki et al., 2016; Takahashi et al., 2012; Xu et al., 2014) combining it with post-mortem tissue analysis to show that radially coherent diffusion signal corresponded to radial glial fibres in the early prenatal period, transitioning to cortico-cortical fibres around 30 weeks, coinciding with the appearance of astrocytes (Takahashi et al., 2012; Xu et al., 2014). However, with whole-tract average values, it is not possible to establish the precise effect of different neurodevelopmental processes on diffusion metrics across gestation. To address this ambiguity, we characterised the entire trajectory of tissue composition changes between the thalamus and the cortex. We found that age-related changes in the tissue and fluid fraction along the tracts concurred with histological observations (Kostović and Judas, 2010). During the early prenatal period (22–25.5 GW), neuronal precursors migrate along the radial glial scaffold from proliferative zones to their destination in the cortical plate and thalamocortical axons accumulate in the superficial subplate, entering a ‘waiting phase’, forming transient synaptic connections (Ghosh et al., 1990; Kostovic and Rakic, 1984; Kostovic and Rakic, 1990). In terms of the diffusion signal, this strongly aligned microstructure of the radial glia is represented in our results by a higher tissue fraction in the transient compartments containing the most migratory cells (such as the VZ, IZ) (Kostović and Judas, 2010). Conversely, we observe the lowest tissue fraction in the early prenatal SP, which predominantly contains hydrophilic extracellular matrix, as demonstrated by rodent and non-human primate studies (Allendoerfer and Shatz, 1994; Miller et al., 2014; Molnár and Hoerder-Suabedissen, 2016). By the mid prenatal period (26–31.5 weeks), we observe increased tissue fraction in the cortical plate, coinciding with the innervation of the cortical plate by thalamocortical axons, increasing soma volume and dendritic branching of CP neurons and CP synaptogenesis (Huttenlocher, 1979; Mrzljak et al., 1992; Huttenlocher and Dabholkar, 1997). We also observe increased tissue fraction in the SP zone in the mid prenatal period, consistent with histological observations of increased coherence of axonal fibres between cortical areas (Takahashi et al., 2012; Xu et al., 2014). The tissue fraction in the VZ and IZ decreases compared to the early prenatal period, corresponding to the timeframe when the radial glial scaffold dissipates (Kinney et al., 1988; Back et al., 2001; Haynes et al., 2005). From the mid to late prenatal period, there is a marked increase in tissue fraction in last third of the axis between thalamus and cortex. By this point in development, the radial glia have converted into oligodendrocyte precursor cells which ensheath the axonal fibres to commence pre-myelination, enhancing the structural integrity of the fibre pathways (Back et al., 2001; Back et al., 2002; Haynes et al., 2005; Kinney et al., 1988; Kinney et al., 1994). A previous study in perinatal rabbits has shown that this oligodendrocyte lineage progression correlates with diffusion metrics (Drobyshevsky et al., 2005), suggesting it is likely to contribute to the increased tissue fraction we observe in the late prenatal period. The tissue fraction increase in the CP area is consistent in time with the lamination of the CP, the elaboration of thalamocortical terminals in layer IV, and a rapid growth of basal dendrites of layer III and V pyramidal neurons (Kostović and Jovanov-Milosević, 2006; Krsnik et al., 2017; Molliver et al., 1973). These high tissue fraction values at the origin and termination of the tracts suggest co-maturation between ascending and descending pathways between the thalamus and cortex to eventually form continuous, structurally mature fibre bundles. This concept was proposed in the 1990s by Blakemore and Molnar, termed the ‘handshake hypothesis’. They suggested that thalamocortical pathways ascending through the internal capsule project to their cortical targets with assistance from reciprocal descending cortical pathways (Molnár and Blakemore, 1995). We hypothesise that continuing this analysis over subsequent weeks into the neonatal period would lead to an increasing tissue fraction in the middle of the axis, as fibre bundles become more uniformly structurally mature and the subplate completely resolves (Kinney et al., 1988; Haynes et al., 2005; Kostović and Jovan