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Synergy between Hoxa1 and Hoxb1: the relationship between arch patterning and the generation of cranial neural crest

Anthony Gavalas, Paul Trainor*, Linda Ariza-McNaughton and Robb Krumlauf{ddagger}

Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
* Present address: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA



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  		Fig. 1. A territory with new characteristics, rx, is formed in the place of presumptive r4 in the Hoxa1-/-/Hoxb1 3'RARE-/-embryos. Wild-type (a,d,g), Hoxa1-/- (b,e,h) and Hoxa1-/-/Hoxb1 3'RARE-/- (c,f,i) embryos were processed for whole-mount in situ hybridisation with kr/Krox20 (a-c), Hoxb2 (d-f) and Hoxa2 (g-i) antisense riboprobes. Double staining with kr/Krox20 shows that the r4 territory of wild-type embryos defined by non-expressing cells (vertical bar in a) was significantly reduced but not eliminated, to similar extents in both Hoxa1-/- and Hoxa1-/-/Hoxb1 3'RARE-/- (vertical bars in b,c, respectively). Hoxb2 expression is continuous throughout the r3-r6 region in both wild-type and Hoxa1-/- embryos (d,e) but not in Hoxa1-/-/Hoxb1 3'RARE-/- embryos (f). There are Hoxb2 non-expressing cells intermingled with r3 cells in both Hoxa1-/-and Hoxa1-/-/Hoxb1 3'RARE-/-embryos (arrowheads in e,f). Expression of Hoxa2 is continuous throughout the r2-r6 region in wild-type (g) as well as Hoxa1-/- (h) and Hoxa1-/-/Hoxb1 3'RARE-/- (i) embryos.

 


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  		Fig. 2. Gradual loss of neural crest markers and reduction of the second arch mesenchymal population. Wild-type (a,d,g,j,m), Hoxa1-/- (b,e,h,k,n) and Hoxa1-/-/Hoxb1 3'RARE-/- (c,f,i,l,o) embryos were processed for whole-mount in situ hybridisation with Hoxb2 (a-c), Hoxa2 (d-f), twist (g-i), Sox10 (j-l) antisense riboprobes and histological staining of paraffin sections (m-o). (a-f) Expression of Hoxb2 and Hoxa2 was significantly reduced in the second pharyngeal arch (pa2) of Hoxa1-/- embryos (b,e) and completely lost in that of Hoxa1-/-/Hoxb1 3'RARE-/- embryos (c,f). Expression of Hoxb2 in the hindbrain of wild-type and Hoxa1-/-embryos was continuous but discontinuous in the hindbrain of Hoxa1-/-/Hoxb1 3'RARE-/- embryos, suggesting the presence of a territory distinct from r3 and r5/r6 (noted with white arrowheads in c). (g-i) Expression of the mesenchymal ncc marker twist was reduced in the second pharyngeal arch of Hoxa1-/- embryos and was lost in that of Hoxa1-/-/Hoxb1 3'RARE-/-embryos. (j-l) The neurogenic ncc marker Sox10 was expressed in the proximal ganglia of wild-type embryos (arrows in j). Its expression was reduced in the facioacoustic ganglion of Hoxa1-/- embryos (arrow in k) and was lost in the more posterior ncc-derived ganglia of Hoxa1-/- embryos (asterisks in k) and in all the ncc-derived ganglia of Hoxa1-/-/Hoxb1 3'RARE-/- embryos (asterisks in l). (m-o) The second pharyngeal arch mesenchymal population was reduced in the Hoxa1-/- embryos and further diminished but not extinguished in the Hoxa1-/-/Hoxb1 3'RARE-/- embryos. ov, otic vesicle; Vg, trigeminal ganglion.

 


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  		Fig. 3. Mitotic and cell death patterns in the second pharyngeal arch are not altered despite the loss of ncc. Wild-type (a,d), Hoxa1-/- (b,e) and Hoxa1-/-/Hoxb1 3'RARE-/- (c,f) embryos were processed for whole-mount immunohistochemical detection of mitosis (a-c) and cell death (d-f). (a-c) Reduced but considerable mitotic activity was still detected in the second pharyngeal arch (pa2) of both Hoxa1-/- and Hoxa1-/-/Hoxb1 3'RARE-/- embryos. (d-f) There was no increase of cell death in the second pharyngeal arch of either Hoxa1-/- or Hoxa1-/-/ Hoxa1-/- Hoxb1 3'RARE-/- embryos.

 


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  		Fig. 4. Signalling molecules are correctly expressed in the second pharyngeal arch despite the loss of ncc. Wild-type (a,d,g,j), Hoxa1-/- (b,e,h,k) and Hoxa1-/-Hoxb1 3'RARE-/- (c,f,i,l) embryos were processed for whole-mount in situ hybridisation with Fgf3 (a-c), Fgf8 (d-f), Shh (g-i) and Bmp7 (j-l) antisense riboprobes. (a-c), Expression of Fgf3 in the first and second pharyngeal pouches (pp1 and pp2, respectively) was not changed in either Hoxa1-/- or Hoxa1–/-/Hoxb1 3'RARE-/- embryos (arrows). (d-f) The weak expression of Fgf8 on the second pharyngeal arch (pa2) was not abolished in either Hoxa1-/- or Hoxa1-/-/Hoxb1 3'RARE-/- embryos (arrows). (g-i) Expression of Shh in the second pharyngeal arch (pa2) was not abolished in either Hoxa1-/- or Hoxa1-/-/Hoxb1 3'RARE-/- embryos (arrows in). (j-l) Similarly, expression of Bmp7 in the second pharyngeal arch (pa2) was not abolished in either Hoxa1-/- or Hoxa1-/-/Hoxb1 3'RARE-/-embryos. ov, otic vesicle.

 


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  		Fig. 5. Pharyngeal pouches and epibranchial placodes develop normally despite the loss of the second arch ncc. Wild-type (a,d), Hoxa1-/- (b,e) and Hoxa1-/-/Hoxb1 3'RARE-/-(c,f) embryos were processed for whole-mount in situ hybridisation with Pax1 (a-c) and Ngn2 (d-f) antisense riboprobes. (a-c) Expression of Pax1 in the first and second pharyngeal pouches (pp1 and pp2 respectively) was not changed in either Hoxa1-/- or Hoxb1 3'RARE-/- embryos (arrows). (d-f) Expression of Ngn2 in the second arch (pa2) geniculate (ge) and more posterior petrosal (pe) and nodose (no) ganglia revealed that their initial stages of development were not affected in either Hoxa1-/- or Hoxa1-/-/Hoxb1 3'RARE-/- embryos (arrows).

 


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  		Fig. 6. Lineage analysis of the r4 cells confirms the lack of migrating ncc in the Hoxa1-/-/Hoxb1 3'RARE-/- mutants. Diagrammatic representation of the experimental procedure (a-c) and the results (d-i). Wild-type (a,d,g), Hoxa1-/- (b,e,h) and Hoxa1-/-/Hoxb1 3'RARE-/- (c,f,i) embryos are labelled with DiI at the presumptive r4 or rx territory (arrowheads in d-f) at the five-somite stage, left in culture for 30-32 hours and examined under fluorescence for ncc migratory patterns (arrows in d,e,g,h). The area of pa2 is shown in g-i under high-power magnification. Ncc migration is reduced in the Hoxa1-/- embryos and completely abolished in Hoxa1-/-/Hoxb1 3'RARE-/- embryos. The segmentation defects of Hoxa1-/- or Hoxa1-/-/Hoxb1 3'RARE-/- result in a less conspicuous preotic sulcus (pos in a-c) and an imperceptible otic sulcus (os in a-c). Brackets in b,e denote the area of the neuroepithelium that was labelled with DiI; brackets in d-f denote the pa2 neural crest migration domain. pa1 and pa2, pharyngeal arches 1 and 2, respectively.

 


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  		Fig. 7. Reduction of second pharyngeal arch delaminating ncc in the Hoxa1-/- and its loss in the Hoxa1-/-/Hoxb1 3'RARE-/- and Hoxa1-/-/Hoxb1-/- mutants. Wild-type (a,b), Hoxa1-/- (c,d), Hoxa1-/-/Hoxb1 3'RARE-/- (e,f) and Hoxa1-/-/Hoxb1-/- (g,h) Haematoxylin/Eosin stained paraffin sections (a,c,e,g) or CRABPI antisense riboprobe stained vibratome sections (b,d,f,h) at the dorsal hindbrain level. Delaminating ncc of the VII and IX ganglia can be seen in the wild-type hindbrains (arrowheads in a,b) anteriorly and posteriorly, respectively, of the otic vesicle (ov). The delaminating population anterior of the otic vesicle is greatly reduced in the Hoxa1-/- mutants (arrowheads in c,d) and completely lost in the Hoxa1-/-/Hoxb1 3'RARE-/- (e,f) and Hoxa1-/-/Hoxb1-/- mutants (g,h) (asterisks). The IXg ncc population still delaminates in all the mutants examined (arrowheads in c-h).

 


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  		Fig. 8. Transplantation experiments demonstrate that the neural crest defect in the Hoxa1-/-/Hoxb1 3'RARE-/- mutants is cell-autonomous. Diagrammatic representation (a,b) of the experimental procedure and the results (c,d). DiI-labelled wild-type r4 cells were transplanted in the r4 of wild-type (a,c) or rx ofHoxa1-/- /Hoxb1 3'RARE-/- (b,d) recipient embryos at the five-somite stage. The embryos were cultured for 24 hours and then observed under fluorescence for migration patterns. The wild-type cells are able to migrate correctly into the second pharyngeal arch (pa2) of both wild-type (c) and Hoxa1-/- /Hoxb1 3'RARE-/- (d) embryos, demonstrating that the ncc defect in the latter is cell-autonomous. os, otic sulcus; pos, preotic sulcus.

 

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© The Company of Biologists Ltd 2001