A spatial-temporal map of glutamatergic neurogenesis in the murine embryonic cerebellar nuclei uncovers a high degree of cellular heterogeneity

Abstract

The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of cerebellar nuclei processing and integration. Nevertheless, our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a mouse genetic inducible fate-mapping study characterizing rhombic lip-derived glutamatergic neurons of the nuclei, the most conspicuous family of long-range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. In mice, they are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral-to-medial progression. While some markers label a heterogeneous population of neurons sharing a common location (BRN2), others appear to be lineage specific (TBR1, LMX1a, and MEIS2). A comparative analysis of TBR1 and LMX1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings contribute to laying the groundwork for a comprehensive functional analysis of nuclear neuron subpopulations.

Keywords: cerebellar nuclei; cerebellum; genetic inducible fate mapping; glutamatergic projection neurons; neurogenesis.

FIGURE 1

Atoh1 lineage progenitors migrate into the embryonic CN from TamE9.5 to TamE12.5, in a lateral‐to‐medial progression. The perimeters of cerebella and each CN are delimited by white dashed and red dotted lines, respectively. (a–e) Anti‐tdT immunofluorescence in rostral hemisections of E18.5 cerebella. Atoh1 lineage neurons are visible in the CN from TamE9.5 to TamE12.5. Only a small number of TamE9.5 neurons of the early‐born cohort are detected (a) and mostly occupy the dorsolateral area of the LAT. TamE10.5 (b) and TamE11.5 (c) tdT+ neurons occupy the dorsolateral portion of the LAT, and the dorsomedial area of the INT and the MED. Solid line box in (b) shows small and large glutamatergic neurons (arrowheads and arrows, respectively). TamE12.5 tdT+ neurons (d) are visible in the medial area of the INT and the entire MED, while only few neurons occupy the lateral area of the LAT. TamE13.5 tdT+ neurons (e) are not visible in the CN. (a′–e′) Anti‐tdT immunofluorescence in caudal hemisections of E18.5 cerebella. Atoh1 lineage neurons are visible in the CN (dotted red lines) from TamE9.5 to TamE12.5. Only sparse neurons occupy the CN in the caudal region of the TamE9.5 cerebellum (a′). TamE10.5 (b′) and TamE11.5 (c′) tdT+ neurons are visible in the MED, INT, and LAT. TamE12.5 tdT+ neurons (d′) are mostly present in the medial area of the INT and the MED. Virtually no TamE13.5 tdT+ neurons (e′) are present in the CN. Solid red arrowheads in (e, e′) point to GC progenitors in the external granular layer. Size bar in (a): 200 μm.

FIGURE 2

The distribution of Atoh1 lineage cells in the adult reflects the lateral to medial progression observed in late embryonic sections. The perimeter of each CN is delimited by dashed lines. Immunostaining against tdT in Atoh1::CreER ^T2 /Rosa26 ^tdT adult (P45) rostral cerebellar hemisections shows recombination in the LAT (cyan dashed lines); (a–c) (magnification in a′–c′). In rostral sections, anti‐tdT staining reveals Atoh1 lineage neurons in the LAT (cyan dashed lines) from TamE10.5 to TamE12.5. The number of tdT‐tagged cells in the LAT decreases from TamE10.5 (a, a′) to TamE12.5 (c, c′). In caudal sections (d–f, magnification in d′–f′), anti‐tdT staining shows Atoh1 lineage neurons in the LAT, INT, and MED (cyan dashed lines). From TamE10.5 (d, d′) to TamE12.5 (f, f′), the number of cells decreases in the LAT and progressively increases in the MED. Crus1, Crus 1 of the ansiform lobule; Crus2, Crus 2 of the ansiform lobule; DC, dorsal cochlear nucleus; FI, flocculus; Int, interposed nucleus; Lat, lateral nucleus; Med, medial nucleus; PFI, paraflocculus; Sim, simple lobule; VCP, Ventral cochlear posterior nucleus; 3Cb, cerebellar lobule III; 4&5Cb, cerebellar lobules IV and V. Size bar in (a): 1 mm.

FIGURE 3

Atoh1‐lineage glutamatergic neurons express BRN2 and MEIS2 in selective territories of the cerebellar nuclei. (a–d) Anti‐tdT and BRN2 double immunofluorescence in rostral hemisections of E18.5 cerebella. BRN2+ cells form compact cellular clusters throughout the LAT and INT. Sparse TamE9.5 tdT+ neurons are also positive for BRN2 in the LAT (solid line box, in a) and INT (a). Most of the TamE10.5 tdT+ neurons are BRN2+ in the dorsolateral area of the LAT (dashed box in b) and in the dorsal area of the INT (dotted box in b). TamE11.5 and TamE12.5 tdT+ neurons are also BRN2+ in the lateral portion of the LAT (dashed boxes in c, d) and dorsomedial portion of the INT (dotted boxes in c, d). (a′–d′) Anti‐tdT and BRN2 double immunofluorescence in caudal hemisections of E18.5 cerebella. Virtually, no tdT+ cells are present in TamE9.5 caudal sections (a′). Only sparse cells are double positive in the MED and INT in TamE10.5 sections (b′, dotted and dashed boxes, respectively). Nearly all tdT+ cells tagged at E11.5 and E12.5 are negative for BRN2 (boxes in c′ and d′). (e–g) Anti‐tdT and MEIS2 double immunofluorescence in rostral hemisections of E18.5 cerebella. Few MEIS2, tdT double‐positive cells are visible in the lateral area of the LAT and INT in TamE10.5 sections (solid box in e). The bulk of MEIS2+ neurons are tagged at E11.5 in the dorsomedial and in the lateral area of the LAT and the dorsolateral area of the INT (solid box in f). MEIS2+ neurons tagged at E12.5 are found in the LAT (solid box in g) and the INT. (e′–g′) Anti‐tdT and MEIS2+ double immunofluorescence in caudal hemisections of E18.5 cerebella. Most of the MEIS2+ are TdT+ in the MED (dashed boxes in e′, f′, g′) both in the middle portion and in the DLP. Size bar in (g′): 200 μm.

FIGURE 4

TBR1+ and LMX1a+ glutamatergic neurons overlap only partially in the MED. The perimeter of each CN is delimited by dotted lines. (a–d′) All sections (E18.5) were immunostained for tdT (red) and TBR (green). (a–d) In rostral sections, TBR1+ cells are located in the ventrolateral and middle area of the MED (solid line boxes). No TBR1+ cells can be found in the INT or LAT (dashed line boxes). (a′–d′) In caudal sections, TBR1+ neurons are localized in the ventrolateral and middle areas of the MED (solid line boxes). (e) Graphical representation of rostral and caudal sections of the MED: Individual TBR1+ neurons shown in (a–d′) are mapped. The map confirms the presence of TBR1+ cells in the ventrolateral and middle area of the MED, and their near absence in the DLP. (f–h′) All sections (E18.5) were immunostained for tdT (red) and LMX1a (green). (f–h) In rostral sections, LMX1a+ cells localize preferentially in the middle area of the MED (solid line boxes). (f′–h′) In caudal sections, LMX1a immunostaining is visible throughout the MED, including the DLP (solid line boxes). (i) Graphical representation of rostral and caudal sections of the MED. Individual LMX1a+ neurons are mapped. The map confirms that LMX1a‐positive cells localize mostly in the middle area and the dorsolateral protuberance of the MED. (f–h′) Presumptive LMX1a+ unipolar brush cells are found outside the CN (e.g., white arrowheads in f′). Size bar in (d′ and h′): 200 μm.

FIGURE 5

LMX1a+ and TBR1+ cell populations are only partially overlapping. (a, b) LMX1a and TBR1 immunostaining in rostral (a) and caudal (b) sections of the E18.5 MED reveals double‐positive neurons, as well as cells that are only positive for either marker. LMX1a–/TBR1+ cells are displayed in boxes 1 and 3 (red arrowheads), LMX1a+/TBR1– cells are displayed in boxes 2 and 4 (green arrowheads), and LMX1a+/TBR1+ cells are displayed in boxes 1 and 3 (yellow arrowheads). Size bar in (b): 200 μm.

FIGURE 6

Graphic representation of the GIFM in CN neurons at different stages of Tam induction in combination with different molecular markers. The last column is the superimposition of the three stages analyzed. (a) In rostral sections, tdT+/BRN2+ neurons are located in the dorsomedial area of the INT and in the dorsolateral part of the LAT. Only a small number of double‐positive neurons is visible in caudal sections of the MED. (b) In rostral sections, tdT+/MEIS2+ neurons mapped the dorsolateral area of the LAT and the dorsal area of the INT. In caudal sections, double‐positive neuron cells are present across the entire MED. (c, d) tdT+/TBR1+ and tdT+/LMX1a+ neurons are visible only in the MED. (c) In caudal sections, most of the tdT+/TBR1+ cells are born between E11.5 and E12.5 and occupy the middle and ventrolateral portions of the MED. (d) In frontal sections, the bulk of tdT+/LMX1a+ cells are born at E12.5 in the medial portion of the MED, while in caudal sections, double‐positive cells are found throughout the MED, including the DLP. (e) The last row represents the superimposition of all tdT+/marker+ cells for each tamoxifen induction within the CN. INTc, caudal interpositus nucleus; INTr, rostral interpositus nucleus; LATc, caudal lateral nucleus; LATr, rostral lateral nucleus; MEDc, caudal medial nucleus; MEDr, rostral medial nucleus.