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First published online May 1, 2006
doi: 10.1242/10.1242/dev.02364

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A threshold requirement for Gbx2 levels in hindbrain development

Laboratory of Cancer and Developmental Biology, NCI-Frederick, National Institutes of Health, Frederick, MD 21702, USA.

View larger version (112K): [in a new window]   Fig. 1. Midline defects in the cerebellum and cerebellar primordium of Gbx2neo homozygotes. (A-C) Dorsal view of posterior E17.5 brains. The cerebellar midline (asterisks) in Gbx2neo homozygotes (B) is absent compared with control (A), whereas the entire cerebellum is absent in Gbx2hb homozygotes (C). Insets in A,B show whole-mount ISH of Math1 expression at E11.5. In controls, Math1 is expressed in proliferative granule cell precursors in the cerebellar primordium (CEP; black arrow), including the vermis primordium (white arrowhead, A inset). Math1 expression is downregulated throughout the CEP and is absent along the vermis primordium of Gbx2neo homozygotes (inset in B). (D,E) Hematoxylin- and Eosin-stained coronal sections of E14.5 cerebella. The vermis primordium is greatly reduced in size and the vermal EGL is much thinner in Gbx2neo homozygotes (E) compared with controls (D). (F,G) Darkfield images of anti-Phospho-Histone H3 staining (white) on coronal sections of E14.5 cerebella. Mitotically active granule cell precursors are greatly reduced throughout the vermal EGL of Gbx2neo homozygotes (G). Insets in F,G show whole-mount ISH of Math1 expression at E13.5, illustrating the loss of midline granule cell precursors (arrowhead). Cb, cerebellum; Cp, choroid plexus; Mb, midbrain; Md, medulla; hEGL, hemispheric external germinal layer; ic, inferior colliculus; vEGL, vermal external germinal layer.

View larger version (51K): [in a new window]   Fig. 2. Rhombomere 2 and its derivatives are absent in Gbx2 hypomorphic mutants. (A-H) Whole-mount ISH using the probe and developmental stage indicated. (I,J) Whole-mount immunohistochemistry using an anti-neurofilament antibody (2H3). A,B,G-J are lateral views; C-F are dorsal views. Krox20 expression occurs normally in r3 and r5 of control (A) and mutant (B) embryos, but the unstained r2 region between the Krox20 and Fgf8 domains is absent in mutant embryos. Cyp26c1 is overstained to demonstrate that r2-specific expression is absent in mutants (D), but evident in r2 and r4 of control embryos (C). Lateral Cyp26c1 staining is due to mesenchymal expression. The anterior limit of Hoxa2 expression in mutants ends at r3 (F), demonstrating that the normal Hoxa2 r2 domain (E) is absent. Krox20-specific expression in cranial nerve (V) boundary cap cells is absent in mutants (H) compared with controls (G). (I) Visualization of cranial nerve and ganglia patterning in a control embryo. (J) In mutants, neural crest-derived sensory neurons connecting the ganglion of the cranial nerve (V) to the hindbrain are absent, and the mandibular branch (N5a) extending into the first branchial arch fails to develop. III, oculomoter; IV, trochlear; V, trigeminal; VII, facial; VIII, vestibulocochlear; d, distal; N5a, mandibular branch of cranial nerve (V); N5m, maxillary branch of cranial nerve (V); N5o, ophthalmic branch of cranial nerve (V); ov, otic vesicle; p, proximal.

View larger version (42K): [in a new window]   Fig. 3. The neo-r cassette causes the production of a hybrid Gbx2 transcript and a reduction of wild-type Gbx2 transcripts in Gbx2neo homozygotes. (A) Schematic representation of the Gbx2neo allele and the hybrid Gbx2neo transcript produced by aberrant splicing into and out of the neo-r cassette. Open arrows indicate the transcriptional direction of Gbx2 and neo-r. Arrowheads indicate the location of primers used for RT-PCR. Larger boxes indicate exons (hatched, non-coding; red, coding; HB, homeobox). Rectangles indicate loxP sites. Sequences surrounding the splice junctions are indicated (red, Gbx2; gray, neo-r). (B) RT-PCR amplifies both wild-type and hybrid Gbx2 transcripts only in Gbx2neo homozygotes. (C) Whole-mount ISH demonstrates a qualitative reduction of Gbx2 expression at E7.75 and 12-13 ss. (D) Quantitative RT-PCR illustrating that wild-type levels of Gbx2 transcript are reduced to 6-10% of normal in Gbx2neo homozygotes at 6-8 ss and 24 ss. y-axis, number of Gbx2 transcripts (log 10); x-axis, genotype (–rt, control samples without reverse transcription).

View larger version (83K): [in a new window]   Fig. 4. Expression of the hybrid Gbx2 transcript recapitulates wild-type Gbx2 expression. (A-H) Lateral views of embryos (A-E) or dorsal views of the cerebellar primordium (CEP; F-H) of the genotype and developmental stage indicated stained by whole-mount ISH for the wild-type Gbx2 (A,C,F) or hybrid transcript (B,D,E,G,H). Gbx2 hybrid mRNA (G,H) and wild-type Gbx2 mRNA (F) are similarly expressed throughout the CEP. Note the increased expression of the hybrid transcript in Gbx2neo homozygotes (H) compared with Gbx2neo heterozygotes (G), and its absence in wild-type embryos (E).

View larger version (90K): [in a new window]   Fig. 5. Mid/hindbrain markers in Gbx2neo homozygotes extend caudally during early somite stages. (A-F) Lateral views of 9-10 ss embryos of the indicated genotype analyzed by whole-mount ISH for the expression of Otx2 (A,B), Fgf8 (C,D) and Wnt1 (E,F).

View larger version (105K): [in a new window]   Fig. 6. Abnormal mid/hindbrain gene expression in mid-gestation Gbx2neo homozygotes. (A-P) Dorsal views of E10.5 embryos of the indicated genotype probed by whole-mount ISH for the expression of Fgf8 (A,B), Fgf17 (C,D), En2 (E,F), En1 (G,H), Otx2 (I,J), Wnt1 (K,L), Spry1 (M,N) and Spry4 (O,P). Insets in A and B show lateral views of E10.5 embryos stained for Fgf8 expression. Asterisks indicate a region of ectopic expression along the dorsal midline of the isthmus and r1.

View larger version (77K): [in a new window]   Fig. 7. The anterior region of r1 in Gbx2neo homozygotes assumes isthmus-like characteristics. (A,B,E-H) Sagittal sections of E10.5 embryos (indicated by white lines in C and D). (C,D,I,J) Dorsal views of E10.5 mid/hindbrain regions. (K,L) Flatmounts of E10.5 lateral mid/hindbrain. (A,B) Normal isthmic Fgf8 expression (A) extends caudally into r1 in Gbx2neo homozygotes (B) (also compare with Fig. 6A,B). (C-F) Math1 expression is absent throughout medial r1 and anterior rhombic lip regions in Gbx2neo homozygotes. (G,H) Immunohistochemical staining using an anti-phospho-Histone H3 antibody reveals that the mitotically less active region of the isthmus r1 in control embryos (G) extends caudally in Gbx2neo homozygotes (H). Red brackets indicate regions of reduced proliferation. (I-L) E10.5 embryos stained for cyclin D2 expression demonstrate that a region of reduced cyclin D2 expression (line in I and right brackets in J-L) is expanded caudally in mutants. c, caudal; CEP, cerebellum primordium; Mb, Midbrain; r, rostral; RL, rhombic lip; r1, rhombomere 1.