Evidence of disrupted rhombic lip development in the pathogenesis of Dandy-Walker malformation

Abstract

Dandy-Walker malformation (DWM) and Cerebellar vermis hypoplasia (CVH) are commonly recognized human cerebellar malformations diagnosed following ultrasound and antenatal or postnatal MRI. Specific radiological criteria are used to distinguish them, yet little is known about their differential developmental disease mechanisms. We acquired prenatal cases diagnosed as DWM and CVH and studied cerebellar morphobiometry followed by histological and immunohistochemical analyses. This was supplemented by laser capture microdissection and RNA-sequencing of the cerebellar rhombic lip, a transient progenitor zone, to assess the altered transcriptome of DWM vs control samples. Our radiological findings confirm that the cases studied fall within the accepted biometric range of DWM. Our histopathological analysis points to reduced foliation and inferior vermian hypoplasia as common features in all examined DWM cases. We also find that the rhombic lip, a dorsal stem cell zone that drives the growth and maintenance of the posterior vermis is specifically disrupted in DWM, with reduced proliferation and self-renewal of the progenitor pool, and altered vasculature, all confirmed by transcriptomics analysis. We propose a unified model for the developmental pathogenesis of DWM. We hypothesize that rhombic lip development is disrupted through either aberrant vascularization and/or direct insult which causes reduced proliferation and failed expansion of the rhombic lip progenitor pool leading to disproportionate hypoplasia and dysplasia of the inferior vermis. Timing of insult to the developing rhombic lip (before or after 14 PCW) dictates the extent of hypoplasia and distinguishes DWM from CVH.

Keywords: Cerebellar vermis hypoplasia; Cerebellum; Dandy–Walker malformation; Development; Rhombic lip.

Fig. 1:. Cerebellar morphobiometry in Dandy-Walker malformation (DWM).

Postnatal MRI through midsagittal plane in normal (a) and DWM (b), with the ‘tail sign’ indicated (white arrowhead). DWM is characterized by cerebellar vermis hypoplasia, a dilated 4^th ventricle (white asterisk) and an enlarged posterior fossa (red arrowhead). Midsagittal section of a 24pcw DWM hindbrain shows characteristic ‘tail sign’ (white arrowhead) and hypoplasia of the vermis (c). Fetal MRI in normal (d) and DWM (e, f) where tegmento-vermian angle (red), cranio-caudal diameter (green) and antero-posterior diameter (yellow) are measured and shown as dashed lines. Measurements for APD, CCD, and TVA in five normal cases (Table 1, grey) and six DWM cases (Table 1, orange) are compared. Data is represented in percentiles and mean +/− standard error of mean.

Fig. 2:. Disproportionate hypoplasia of cerebellar posterior lobules in Dandy-Walker malformation.

Hematoxylin and Eosin (H&E) stained midsagittal sections of the developing normal (a-e) and DWM human cerebellum (f-j). Dots indicate number of lobuli or foliation units present at each timepoint. The anterior, central, and posterior lobes are represented by blue, white, and red dots respectively. Distribution of the number of cerebellar lobuli per age in Control and DWM cerebellum indicates that while the posterior lobe grows at a much slower rate in normals, the posterior lobe remains disproportionately hypoplastic in DWM (k). Number of lobuli by both region and diagnosis in normal control (grey) and DWM (yellow) cerebella indicates disproportionate posterior lobule hypoplasia in DWM (l). KI67 immunohistochemical assays indicate no significant difference in proliferation between normal and DWM in respective lobes (t-test), although proliferation is significantly lesser in the posterior lobes when compared to the anterior lobe within the normal and DW cerebellum. Mean is represented by lines while dots represent individual data points (m). Scale bar = 1mm (a-j).

Fig. 3:. The cerebellum in Dandy-Walker malformation displays a characteristically blunt and obtuse fastigial angle

(a-i) H&E stained midsagittal sections show the progression of the fastigial angle in a normal cerebellum as it changes from obtuse (black dashes) to acute (blue dashes). The fastigial angle is not age-dependent, but dependent on the internalization of the rhombic lip and growth of the posterior lobe around 14pcw (b, c; d, e). (j, k) Cerebellar vermis hypoplasia (CVH) cases possess an acute fastigial angle (red dashes), suggesting the rhombic lip is not disrupted until after internalization. (l, m) DWM cases demonstrate a blunt and obtuse fastigial angle (red dashes). (n) Graph comparing the fastigial angle (y-axis) in DWM (yellow), CVH (yellow), and normal cerebella (grey) in a grouped-column scatterplot. The fastigial angle becomes significantly acute in the normal cerebellum after RL internalization (t-test; p<0.0001). In the DWM cerebellum the fastigial angle is only similar to normal pre-internalization cerebella but remains significantly obtuse compared to normal post-internalization cerebella (t-test; P<0.0001) and CVH whose fastigial angles are acute themselves (p<0.0001; compared to DWM). Mean is represented by lines while dots represent individual data points. Scale bar = 0.5 mm (black) and 1 mm (blue).

Fig. 4:. The ‘Tail Sign’ is a conspicuous feature of Dandy-Walker malformation

Midsagittal MRI scans of postnatal (a) and fetal DWM (b) show a characteristic ‘tail sign’ (red arrowhead) that is also conspicuous during autopsy (c, d) and histological analysis (e-k). LMX1A (j) and Calbindin (k) expression indicate that the tail sign is composed of non-internalized rhombic lip (red arrowhead) attached to a partially formed unpaired posterior lobule. Ventricular zone (blue arrowhead) and fastigial recess (black arrowhead) are also marked (j). Pie charts showing the presence of the tail-sign in the majority (75%) of DWM cases studied (l), and the developmental status of the rhombic lip in each of these same cases, with the rhombic lip being absent in 32% of the cases studied, while an underdeveloped reduced rhombic lip was seen in 46% of cases studied. Only 21% of cases analyzed had an internalized albeit reduced rhombic lip (m). Scale bar = 1 mm (e-k)

Fig. 5:. Histopathology of aberrant rhombic lip development in Dandy-Walker malformation

Immunohistochemistry and in situ hybridization assays data on the human cerebellar rhombic lip (red arrowheads, a-g, j-q). KI67 immunostaining in the RL^VZ (red asterisk) RL^SVZ (yellow asterisk) of normal (a) and DW cerebella (b-d). Graph comparing percentage of Ki67+ cells (y-axis) in the RL^VZ , RL^SVZ and the whole RL (h). SOX2 immunostaining in the RL^VZ , RL^SVZ of normal (e) and DW cerebella (f, g). Graph comparing percentage of SOX2+ cells (y-axis) in the RL^VZ , RL^SVZ and the whole RL (i). Tail-like structure retains rhombic lip identity as evinced by LMX1A ISH (j), PAX6, and TBR2 immunohistochemistry (k, l). (l) is an inset of (k) denoted by a white dashed box. Marked increase in TBR2 staining in the RL^SVZ shows buildup of precociously differentiated UBCs (l). GFAP-KI67 staining in the DWM RL (m). GFAP-CD34 staining for the rhombic lip vascular bed (white arrowheads) indicates its organized arrangement in normal (n) while being displaced and disorganized in the DWM rhombic lip (m, o-q). In Graphs h and i, Mean is represented by lines while dots represent individual data points (t-test; p<0.05). Scale bar = 0.5 mm (black) and 100 um (white).

Fig. 6:. Transcriptional profiling of the Dandy-Walker malformation rhombic lip.

(a) H&E stained midsagittal sections of the DWM cerebellum depicting the rhombic lip progenitor zone isolated by laser-capture microdissection. (b) Scatterplot of the logarithmic fold changes (y-axis) between DWM and control normal rhombic lip versus the mean of normalized counts (x-axis). Genes upregulated in the DWM rhombic lip are shown in green and downregulated are shown in blue. (c) Box plots of notable differentially expressed genes between normal (grey) and DWM (yellow). Gene expression is measured in transcript per million (TPM). (d) Gene ontology (GO) enrichment analysis among differentially expressed genes. The top 10 GO categories are shown. Scale bar = 1mm (a,b)

Fig. 7:. Developmental model for Dandy-Walker malformation in humans

Model for the internalization of the rhombic lip (yellow), and the subsequent sharpening of the fastigial angle (red dashes). During normal development, the rhombic lip compartmentalizes into a RL^VZ (red) and RL^SVZ (dark green) separated by a vascular bed (black). The rhombic lip feeds granule neuronal precursors (light green) into the external granule layer promoting growth of the posterior vermis (dark grey). The rhombic lip also produces unipolar brush cells (purple) that stream into the multiple cerebellar lobules. During early development the RL extends beyond the perimeter of the posterior lobule (blue dashes). Outward growth of the posterior-most lobule (black arrowheads) causes the internalization of the rhombic lip and a concomitant blunting of the fastigial angle (red dashes). In the DWM cerebellum, insults such as vascular disruptions result in reduced self-renewal and premature differentiation of rhombic lip progenitors (11–16pcw, magenta), which further leads to reduced growth of the posterior vermis, resulting in the fastigial angle remaining obtuse. Prematurely differentiated UBCs build up in the DWM rhombic lip (16–20pcw, purple). Reduced growth of the posterior vermis correlates with failure of the rhombic lip to internalize, resulting in tail-like structure (Dandy-Walker, 20pcw, box).