A novel role for Pax6 in the segmental organization of the hindbrain

Abstract

SUMMARYComplex patterns and networks of genes coordinate rhombomeric identities, hindbrain segmentation and neuronal differentiation and are responsible for later brainstem functions. Pax6 is a highly conserved transcription factor crucial for neuronal development, yet little is known regarding its early roles during hindbrain segmentation. We show that Pax6 expression is highly dynamic in rhombomeres, suggesting an early function in the hindbrain. Utilization of multiple gain-and loss-of-function approaches in chick and mice revealed that loss of Pax6 disrupts the sharp expression borders of Krox20, Kreisler, Hoxa2, Hoxb1 and EphA and leads to their expansion into adjacent territories, whereas excess Pax6 reduces these expression domains. A mutual negative cross-talk between Pax6 and Krox20 allows these genes to be co-expressed in the hindbrain through regulation of the Krox20-repressor gene Nab1 by Pax6. Rhombomere boundaries are also distorted upon Pax6 manipulations, suggesting a mechanism by which Pax6 acts to set hindbrain segmentation. Finally, FGF signaling acts upstream of the Pax6-Krox20 network to regulate Pax6 segmental expression. This study unravels a novel role for Pax6 in the segmental organization of the early hindbrain and provides new evidence for its significance in regional organization along the central nervous system.

A New Role of Hindbrain Boundaries as Pools of Neural Stem/Progenitor Cells Regulated by Sox2

“…1A, Figs. 2 and 3; Additional file 1) [15,34,43,72,73]. Notably, DAPI-negative gaps are present in the mantle-most layer of boundaries, which reflect nuclei-free domains (Additional file 1), consistent with previously published data [14][15][16].…”

“…All these proteins are known to be co-expressed with Sox2 in different NSCs [48-50, 61, 77-80]. Notably, Pax6 was previously found to be enriched at chick HBs [15,72]. Immunostaining of embryos revealed the co-localization of these markers with Sox2 within HBs ( Fig.…”

“…Previous studies have shown that Sox2 is broadly expressed in the early chick hindbrain [52,71]. Here, we examined Sox2 expression at later stages (st. [15][16][17][18], when multiple other boundary markers are fully expressed [34,72]. Determination of the boundary regions was made based on the clear morphological bulges at these sites which contain an accumulation of cell bodies at the ventricular side compared to rhombomeres, as shown by DAPI nuclear staining, as well as by the specialized expression of different markers at these regions (Fig.…”

Hox proteins drive cell segregation and non-autonomous apical remodelling during hindbrain segmentation

“…Although the r6/r7 interface is a bona fide lineage restriction (Fraser et al, 1990; Birgbauer and Fraser, 1994), we showed that it is less straight and displays less pronounced apical remodelling than other interfaces. This correlates, in the chick, with weaker Fgf3 and Pax6 expression at r6/r7 than at the Krox20 boundaries and manipulations of either gene at early somite stages can alter segmentation and boundary gene expression (Marín and Charnay, 2000; Weisinger et al, 2012; Kayam et al, 2013). However, when we ectopically expressed Pax6 or Fgf3 (or inhibited Fgf signalling) at the later developmental stage used in our Hox electroporations, apical remodelling at rhombomere boundaries was not detectably altered (data not shown).…”

Conserved localization of Pax6 and Pax7 transcripts in the brain of representatives of sarcopterygian vertebrates during development supports homologous brain regionalization

“…Interestingly, it has been suggested that Pax6 is involved in the specification of subtypes of hindbrain neurons (Osumi et al, 1997) and is currently determined as crucial factor in the segmental organization of the early hindbrain (Kayam et al, 2013). …”