Cell division angle predicts the level of tissue mechanics that tune the amount of cerebellar folding.

Modeling has led to proposals that the amount of neural tissue folding is set by the level of differential expansion between tissue layers and that the wavelength is set by the thickness of the outer layer. Here, we used inbred mouse strains with distinct amounts of cerebellar folding to investigate these predictions. We identified a distinct critical period during which the folding amount diverges between the two strains. In this period, regional changes in the level of differential expansion between the external granule layer (EGL) and underlying core correlate with the folding amount in each strain. Additionally, the thickness of the EGL varies regionally during the critical period alongside corresponding changes in wavelength. The number of SHH-expressing Purkinje cells predicts the folding amount, but the proliferation rate in the EGL is the same between the strains. However, regional changes in the cell division angle within the EGL predicts both the tangential expansion and the thickness of the EGL. Cell division angle is likely a tunable mechanism whereby both the level of differential expansion along the perimeter and the thickness of the EGL are regionally tuned to set the amount and wavelength of folding.

Cell division angle regulates the tissue mechanics and tunes the amount of cerebellar folding.

Modeling has proposed that the amount of neural tissue folding is set by the level of differential-expansion between tissue layers and that the wavelength is set by the thickness of the outer layer. Here we used inbred mouse strains with distinct amounts of cerebellar folding to investigate these predictions. We identified a critical period where the folding amount diverges between the strains. In this period, regional changes in the level of differential-expansion between the external granule layer (EGL) and underlying core correlate with the folding amount in each strain. Additionally, the thickness of the EGL is regionally adjusted during the critical period alongside corresponding changes in wavelength. While the number of SHH-expressing Purkinje cells predicts the folding amount, the proliferation rate in the EGL is the same between the strains. However, regional changes in the cell division angle within the EGL predicts both the tangential-expansion and thickness of the EGL. Cell division angle is likely a tunable mechanism whereby both the level of differential-expansion and thickness of the EGL are regionally tuned to set the amount and wavelength of folding.

Organization of Embryonic Morphogenesis via Mechanical Information.

Embryonic organizers establish gradients of diffusible signaling molecules to pattern the surrounding cells. Here, we elucidate an additional mechanism of embryonic organizers that is a secondary consequence of morphogen signaling. Using pharmacological and localized transgenic perturbations, 4D imaging of the zebrafish embryo, systematic analysis of cell motion, and computational modeling, we find that the vertebrate tail organizer orchestrates morphogenesis over distances beyond the range of morphogen signaling. The organizer regulates the rate and coherence of cell motion in the elongating embryo using mechanical information that is transmitted via relay between neighboring cells. This mechanism is similar to a pressure front in granular media and other jammed systems, but in the embryo the mechanical information emerges from self-propelled cell movement and not force transfer between cells. The propagation likely relies upon local biochemical signaling that affects cell contractility, cell adhesion, and/or cell polarity but is independent of transcription and translation.

Cerebellar folding is initiated by mechanical constraints on a fluid-like layer without a cellular pre-pattern.

Models based in differential expansion of elastic material, axonal constraints, directed growth, or multi-phasic combinations have been proposed to explain brain folding. However, the cellular and physical processes present during folding have not been defined. We used the murine cerebellum to challenge folding models with in vivo data. We show that at folding initiation differential expansion is created by the outer layer of proliferating progenitors expanding faster than the core. However, the stiffness differential, compressive forces, and emergent thickness variations required by elastic material models are not present. We find that folding occurs without an obvious cellular pre-pattern, that the outer layer expansion is uniform and fluid-like, and that the cerebellum is under radial and circumferential constraints. Lastly, we find that a multi-phase model incorporating differential expansion of a fluid outer layer and radial and circumferential constraints approximates the in vivo shape evolution observed during initiation of cerebellar folding.

Genetic deletion of genes in the cerebellar rhombic lip lineage can stimulate compensation through adaptive reprogramming of ventricular zone-derived progenitors.

BACKGROUND: The cerebellum is a foliated posterior brain structure involved in coordination of motor movements and cognition. The cerebellum undergoes rapid growth postnataly due to Sonic Hedgehog (SHH) signaling-dependent proliferation of ATOH1+ granule cell precursors (GCPs) in the external granule cell layer (EGL), a key step for generating cerebellar foliation and the correct number of granule cells. Due to its late development, the cerebellum is particularly vulnerable to injury from preterm birth and stress around birth. We recently uncovered an intrinsic capacity of the developing cerebellum to replenish ablated GCPs via adaptive reprogramming of Nestin-expressing progenitors (NEPs). However, whether this compensation mechanism occurs in mouse mutants affecting the developing cerebellum and could lead to mis-interpretation of phenotypes was not known. METHODS: We used two different approaches to remove the main SHH signaling activator GLI2 in GCPs: 1) Our mosaic mutant analysis with spatial and temporal control of recombination (MASTR) technique to delete Gli2 in a small subset of GCPs; 2) An Atoh1-Cre transgene to delete Gli2 in most of the EGL. Genetic Inducible Fate Mapping (GIFM) and live imaging were used to analyze the behavior of NEPs after Gli2 deletion. RESULTS: Mosaic analysis demonstrated that SHH-GLI2 signaling is critical for generating the correct pool of granule cells by maintaining GCPs in an undifferentiated proliferative state and promoting their survival. Despite this, inactivation of GLI2 in a large proportion of GCPs in the embryo did not lead to the expected dramatic reduction in the size of the adult cerebellum. GIFM uncovered that NEPs do indeed replenish GCPs in Gli2 conditional mutants, and then expand and partially restore the production of granule cells. Furthermore, the SHH signaling-dependent NEP compensation requires Gli2, demonstrating that the activator side of the pathway is involved. CONCLUSION: We demonstrate that a mouse conditional mutation that results in loss of SHH signaling in GCPs is not sufficient to induce long term severe cerebellum hypoplasia. The ability of the neonatal cerebellum to regenerate after loss of cells via a response by NEPs must therefore be considered when interpreting the phenotypes of Atoh1-Cre conditional mutants affecting GCPs.

Cerebellar granule cell replenishment postinjury by adaptive reprogramming of Nestin+ progenitors.

Regeneration of several organs involves adaptive reprogramming of progenitors, but the intrinsic capacity of the developing brain to replenish lost cells remains largely unknown. Here we found that the developing cerebellum has unappreciated progenitor plasticity, since it undergoes near full growth and functional recovery following acute depletion of granule cells, the most plentiful neuron population in the brain. We demonstrate that following postnatal ablation of granule cell progenitors, Nestin-expressing progenitors, specified during mid-embryogenesis to produce astroglia and interneurons, switch their fate and generate granule neurons in mice. Moreover, Hedgehog signaling in two Nestin-expressing progenitor populations is crucial not only for the compensatory replenishment of granule neurons but also for scaling interneuron and astrocyte numbers. Thus, we provide insights into the mechanisms underlying robustness of circuit formation in the cerebellum and speculate that adaptive reprogramming of progenitors in other brain regions plays a greater role than appreciated in developmental regeneration.

Cross-Scale Integrin Regulation Organizes ECM and Tissue Topology.

The diverse morphologies of animal tissues are underlain by different configurations of adherent cells and extracellular matrix (ECM). Here, we elucidate a cross-scale mechanism for tissue assembly and ECM remodeling involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5. Fluorescence cross-correlation spectroscopy within the zebrafish paraxial mesoderm mesenchyme reveals a physical association between Integrin α5 on adjacent cell membranes. This Integrin-Integrin complex correlates with conformationally inactive Integrin. Cadherin 2 stabilizes both the Integrin association and inactive Integrin conformation. Thus, Integrin repression within the adherent mesenchymal interior of the tissue biases Fibronectin fibrillogenesis to the tissue surface lacking cell-cell adhesions. Along nascent somite boundaries, Cadherin 2 levels decrease, becoming anti-correlated with levels of Integrin α5. Simultaneously, Integrin α5 clusters and adopts the active conformation and then commences ECM assembly. This cross-scale regulation of Integrin activation organizes a stereotypic pattern of ECM necessary for vertebrate body elongation and segmentation.

Regulated tissue fluidity steers zebrafish body elongation.

The tailbud is the posterior leading edge of the growing vertebrate embryo and consists of motile progenitors of the axial skeleton, musculature and spinal cord. We measure the 3D cell flow field of the zebrafish tailbud and identify changes in tissue fluidity revealed by reductions in the coherence of cell motion without alteration of cell velocities. We find a directed posterior flow wherein the polarization between individual cell motion is high, reflecting ordered collective migration. At the posterior tip of the tailbud, this flow makes sharp bilateral turns facilitated by extensive cell mixing due to increased directional variability of individual cell motions. Inhibition of Wnt or Fgf signaling or cadherin 2 function reduces the coherence of the flow but has different consequences for trunk and tail extension. Modeling and additional data analyses suggest that the balance between the coherence and rate of cell flow determines whether body elongation is linear or whether congestion forms within the flow and the body axis becomes contorted.