Wnt1 expression temporally allocates upper rhombic lip progenitors and defines their terminal cell fate in the cerebellum

Abstract

The cerebellum (Cb) controls movement related physiology using a diverse array of morphologically and biochemically distinct neurons. During development, the Cb is derived from rhombomere 1 (r1), an embryonic compartment patterned by a signaling center referred to as the isthmus organizer. The secreted glycoprotein WNT1 is expressed in the midbrain primordia (mesencephalon, mes) and at the posterior limit of the mes. WNT1 plays a pivotal role in maintaining the isthmus organizer and mutations in Wnt1 produce severe Cb defects that are generally attributed to aberrant organizer activity. Interestingly, Wnt1 is also expressed at the most posterior limit of dorsal r1, in a region known as the upper rhombic lip (URL). However, the distribution and molecular identity of Wnt1 expressing progenitors have not been carefully described in r1. We used Wnt1-Venus transgenic mice to generate a molecular map of Wnt1 expressing progenitors in relation to other well characterized Cb biomarkers such as MATH1 (ATOH1), LMX1a and OTX2. Our analysis validated Wnt1 expression in the URL and revealed molecularly-defined developmental zones in r1. We then used genetic inducible fate mapping (GIFM) to link transient Wnt1 expression in r1 to terminal cell fates in the mature Cb. Wnt1 expressing progenitors primarily contributed to neurons in deep cerebellar nuclei, granule cells, and unipolar brush cells in distinct but overlapping temporal windows and sparsely contributed to inhibitory neurons and Bergmann glia. We further demonstrate that the Wnt1 lineage does not follow a competency model of progressive lineage restriction to generate the Cb or the functionally related precerebellar system. Instead, progenitors initiate Wnt1 expression de novo to give rise to each Cb cell type and precerebellar nuclei. We also used GIFM to determine how the temporal control of Wnt1 expression is related to molecular identity and cell migration in Cb development. Our findings provide new insight into how lineage and timing establish cell diversity within the Cb system.

Fig. 1. Wnt1 expression in r1

(A–B,E–F,I–J,M–N) ISH revealed Wnt1 and Math1 expression in the URL (arrow) in wild type mice at E11.5 and E12.5. (C–D,G–H,K–L,O–P) ICC with an anti-GFP antibody and indicated biomarkers on sections from Wnt1-Venus embryos uncovered a population of Wnt1 expressing progenitors (green) coexpressing LMX1a (C,G,K,O red) in the posterior URL (white arrow) and OTX2 (D,H,L,P red) in the roof plate (red arrow). Wnt1 expressing progenitors in the anterior URL did not express either of these markers (solid arrowhead). GFP perdurance was used as a transient lineage tracer and reveals Wnt1-derived cells in the lateral migratory stream (open arrowheads). Scale bar: 61 μm.

Fig. 2. Wnt1 expressing progenitors temporally contributed to the DCN

ICC on adult sagittal sections with antibodies that recognized β-gal (red) or GFP (green) revealed that Wnt1 expressing progenitors contributed to all three DCN. (A–C) The Wnt1 lineage marked at E10.5 preferentially populated the interpositus and dentate nuclei. The Wnt1-derived cells in these two nuclei have larger cell nuclei (arrows) than those in the fastigial nuclei (arrowheads). (D–F) The Wnt1 lineage marked at E11.5 represented the peak of DCN marking. Cells with both large (arrows) and small (arrowheads) nuclei are found across all three DCN. (G–I) The Wnt1 lineage marked at E12.5 preferentially populated the fastigial nuclei while the interpositus and dentate had fewer Wnt1-derived cells. The Wnt1-derived cells in the fastigial nuclei had large cell nuclei (arrows) while those in the lateral DCN had small nuclei (arrowheads). Scale bars: 120 μm (A–I), 30 μm (insets).

Fig. 3. The Wnt1 lineage marked at E11.5 primarily contributed to the medial-posterior Cb cortex

ICC on adult sagittal sections with antibodies against β-gal (red) or GFP (green) revealed that Wnt1 expressing cells at E11.5 gave rise to cells in the granular layer. (A,C,D) Medially, Wnt1-derived cells populated every folia, but a substantially greater contribution was seen in folia IX and X. (B,E,F) Laterally, fate mapped cells were evenly distributed across all folia and were less densely populated in the posterior Cb. Scale bars: 1.0 mm (A,B) 61 μm (C–F).

Fig. 4. The Wnt1 lineage marked at E12.5 contributed to the entire Cb cortex

(A–F) ICC on adult sagittal sections with antibodies against β-gal (red) or GFP (green) revealed that Wnt1 expressing cells at E12.5 populated the granular layer of every folia and represented the peak contribution to this region. Wnt1-derived Purkinje cells were also observed (C,D arrows). Scale bars: 1.0 mm (A,B) 61 μm (C–F).

Fig. 5. Unipolar brush cells are derived from the Wnt1 lineage

(A–D) ICC on adult sagittal sections with antibodies against β-gal (red) or Calretinin (CALR, green) uncovered that Wnt1 expressing progenitor cells (red) contributed to unipolar brush cells (green) in the granular layer of folia IX and X (arrows). The Wnt1 lineage from E12.5 (C) made the most substantial contribution. Scale bar: 30 μm.

Fig. 6. Molecular analysis of the Wnt1 lineage in the adult Cb cortex

ICC on adult sagittal sections with antibodies recognizing either β-gal or EGFP and cell type specific biomarkers. (A) Three dimensional rendering of a Wnt1-derived neuron marked at E11.5 (using the mGFP reporter) demonstrated the distinct morphology of a Purkinje cell with both nuclear β-gal (red) and membrane bound GFP (green) immunolabeling. (B) The contribution of the Wnt1 lineage to scattered Purkinje cells was confirmed by Calbindin (CALB, green) labeling. (C) Wnt1 fate mapping with the Z/EG reporter revealed that the Wnt1 lineage (green) marked at E11.5 also contributed to Bergmann glia, identified based on their cell morphology and position between Purkinje cells (CALB, red). (D) Three dimensional rendering of the Wnt1-derived Bergmann glia viewed in the XZ plane showed their close apposition with Purkinje cells. (E–F) Antibody labeling against PAX2 (E, green) or PARV (F, green) showed that the Wnt1 lineage marked at E12.5 contributed to a small population of inhibitory interneurons of the granular layer and molecular layer. Scale bar: 30 μm.

Fig. 7. Wnt1 expressing progenitors

ICC with an antibody against tdTomato (DsRed, red) to detect the Wnt1 lineage marked at the indicated stages and analyzed on E14.5 sagittal sections. (A,B,C) The Wnt1 lineage marked at E10.5 was located in the posterior URL (arrowheads), along the surface of the Cb primordia, and within the anterior extent of the intermediate (B) and lateral (C) Cb (arrows). (C,D,E) The Wnt1 lineage marked at E12.5 was positioned throughout the URL (arrowheads) and along the surface of the Cb primordia. However, progenitors expressing Wnt1 at E12.5 did not populate the anterior Cb. In addition, a few fate mapped cells were seen above the ventricular zone with marking at E12.5 (open arrowheads). choroid plexus (cp), midbrain (mb). Scale bar: 120 μm.

Fig. 8. Wnt1 expressing progenitors differentially migrate away from the URL by E18.5

ICC with an antibody against tdTomato (DsRed, red) to detect the Wnt1 lineage marked at the indicated stages and analyzed on E18.5 sagittal sections. (A,B) The Wnt1 lineage marked at E10.5 was sparsely scattered within the Cb core, anterior URL (arrows, inset shows higher magnification of the URL), and posterior EGL (open arrowheads). Wnt1-derived projections (arrowheads) ran longitudinally in the lateral Cb and transversely in the medial Cb (projections are shown in more detail in Figure S6). (C,D) The Wnt1 lineage marked at E11.5 had a similar distribution, but the extent of labeling was increased versus marking at E10.5. (E,F) The Wnt1 lineage marked at E12.5 constituted the peak of Cb marking and produced an abundance of labeled cells within the URL, Cb core, and EGL at E18.5. (G,H) The Wnt1 lineage marked at E13.5 contributed substantially to the EGL with a posterior bias. choroid plexus (cp), midbrain (mb). Scale bar: 120 μm.

Fig. 9. Wnt1 expressing progenitors distinctively contributed to the developing Cb at P7

ICC with an antibody against tdTomato (DsRed, red) on P7 fate mapped sagittal sections. (A,B) The Wnt1 lineage marked at E10.5 primarily contributed to the Cb core, posterior EGL, and posterior IGL. Wnt1-derived projections (arrowheads) were preferentially located in folia VI–VII and the hemispheres at P7 (C,D) The Wnt1 lineage marked at E11.5 had a similar distribution, but the extent of labeling was increased from marking at E10.5. Wnt1-derived cells populated every folia, but a greater contribution was seen in the posterior vermis. (E,F) The Wnt1 lineage from E12.5 constituted the peak of Cb marking and produced cells within the DCN and Cb cortex. (G,H) The Wnt1 lineage marked at E13.5 contributed substantially to the EGL and Cb cortex with a posterior bias. choroid plexus (cp), midbrain (mb). Scale bar: 250 μm

Fig. 10. Molecular analysis of the Wnt1 lineage in the developing Cb cortex

(A,B,E,F) ICC with antibodies that recognized tdTomato (DsRed, red) or Calretinin (CALR, green) on P7 sagittal sections uncovered that Wnt1 expressing progenitors (red) contributed to unipolar brush cells (green) in folia IX and X. (C,D,G,H) Antibody labeling against PAX2 (green) showed that the Wnt1 lineage gave rise to inhibitory interneurons of the granular layer. (I–P) ICC with Parvalbumin (PARV, green) revealed that the Wnt1 lineage contributed to scattered Purkinje cells (arrows) and Bergmann glia (arrowheads). Scale bar, 61 μm.

Fig. 11. The temporal dissection of the Wnt1 lineage contribution to the precerebellar system

(A,C,E,G,I,K) Sagittal schematics illustrate the section plane used to analyze each precerebellar nuclei. The VN (A), ECN (C), ION (E), PGN (G,K) and LRN (I) are depicted in red and the RTN (G,K) in green. The imaging frame is delineated by the box. (B,D,F,H,J,L) ICC on adult sagittal sections with antibodies against β-gal (red) revealed that Wnt1 expressing progenitors (red) contributed to all six precerebellar nuclei and the magnitude of this contribution shifted across development. (A,B) In the VN, the onset of Wnt1 contribution was at E8.5 with peak marking from E9.5-E11.5; E9.5 is shown. (C,D) In the ECN, the onset of Wnt1 contribution was at E8.5 with a broad peak in marking from E9.5-E13.5; E11.5 marking is shown. (E,F) In the ION, Wnt1-derived neurons were observed with marking at E10.5 and E11.5. (G,H) The PGN had a peak contribution from the Wnt1 lineage marked at E12.5. (I,J) In the LRN, the onset of Wnt1 contribution was at E9.5 with peak marking from E10.5-E13.5. (K,L) The peak contribution to the lateral PGN and RTN was at E12.5. Scale bar: 120 μm.