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First published online 1 February 2006
doi: 10.1242/dev.02267

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Competence of cranial ectoderm to respond to Fgf signaling suggests a two-step model of otic placode induction

Gonda Department of Cell and Molecular Biology, House Ear Institute, 2100 West 3rd Street, Los Angeles, CA 90057, USA.

View larger version (62K): [in a new window]   Fig. 1. Fgf signaling is necessary for the induction but not maintenance of some otic placode markers. (A) A region of chick head between the first pair of somites and the midbrain was isolated from embryos between 0-8 ss and cultured as a chunk in collagen gel in the presence or absence of the Fgf receptor inhibitor SU5402. (B) Chunk cultures stained after 24 hours with antibodies to Pax2, Epha4 or Dlx3 or processed for in situ hybridization with a Bmp7 probe. Ectoderm positive for each marker is shown with an arrow; negative ectoderm is indicated with an arrowhead.

View larger version (52K): [in a new window]   Fig. 2. Fgf signaling is sufficient for expression of most but not all otic placode markers. (A) Ectoderm from the level of the presumptive trigeminal placode was isolated from 0-4 ss chick embryos and cultured in collagen gels for 24 hours in the presence or absence of Fgf2. A dose response curve for Pax2 expression cultured in Fgf2 for 24 hours is shown. (B) Pax2 is detected in explants after 6 hours of culture in collagen gels, but not at 4 hours. (C) Fgf2 (50 ng/ml) induces expression of Pax2, Dlx3, Epha4 but not Bmp7. None of the four markers are expressed in the absence of Fgf2.

View larger version (76K): [in a new window]   Fig. 3. Induction of otic placode markers occurs in the absence of neural or mesodermal markers. Presumptive trigeminal ectoderm was cultured in the presence of 50 ng/ml Fgf2 for between 3 and 24 hours, and examined for the expression of neural and mesodermal markers. Tailless marks the anterior CNS, Krox20 marks rhombomeres 3 and 5, Wnt8c marks rhombomere 4 and the caudal neural plate, while Sax1 exclusively marks the caudal neural plate. Sip1 and Zic2 are pan-neural markers, while brachyury marks early mesoderm. The panels show two controls: the normal expression pattern of each gene in vivo and tissue positive for each marker processed for in situ hybridization in collagen gels. These are compared with the expression seen in cultured epiblast +Fgf2 after 24 hours. Epiblast explants cultured in the presence of Fgf2 were completely negative for all markers at all time points examined (Table 3).

View larger version (50K): [in a new window]   Fig. 4. Different regions of ectoderm are differentially responsive to Fgf2. (A) Ectoderm was taken from the anterior epiblast of HH stage 3+-4 chick embryos, or from the presumptive trigeminal, or presumptive otic ectoderm, or lateral to the presumptive trigeminal ectoderm of 0-4 ss embryos. Cultures were grown in 50 ng/ml Fgf2 for 24 hours. (B) Pax2 is expressed in presumptive otic or presumptive trigeminal level ectoderm, but not ectoderm taken from lateral regions or from the anterior epiblast. (C) Histogram showing Pax2 induction in different ectoderm populations in the presence or absence of Fgf2. Numbers above the columns indicate the number of explants tested in each case. (D) Sections of 2 ss chick embryos through the region of the presumptive trigeminal placode. Embryos were processed with an in situ probe for Eya2, or with an antibody that recognizes all Distalless proteins (pan-Dll). Expression of pre-placodal markers is seen in ectoderm adjacent to the neural plate (black arrow or black and white brackets), but not in lateral ectoderm (red arrow and brackets).

View larger version (45K): [in a new window]   Fig. 5. Upregulation of pre-placodal markers in anterior epiblast by grafting to the pre-placodal region. (A) Anterior epiblast was isolated from HH stage 3+-4 quail embryos and grafted into chick hosts at 0-4 ss. Grafts were placed either adjacent to the neural plate at the level of the presumptive trigeminal placode, or lateral to the presumptive trigeminal placode. (B) Embryos were examined after 4 or 8 hours with a pan-Distalless antibody (pan-Dll) or an Eya2 probe, together with QCPN antibody to label quail tissue. Grafts adjacent to the neural plate (trigeminal) express both pan-Dll and Eya2 after 8 hours. Grafts lateral to the neural plate do not express either marker after 4 or 8 hours.

View larger version (40K): [in a new window]   Fig. 6. Pre-placodal markers are upregulated following grafting adjacent to the anterior-most neural plate, but not the trunk neural plate. (A) Anterior epiblast was isolated from HH stage 3+-4 quail embryos and grafted into chick hosts of between 0-4 ss at the level of either the most anterior part of the pre-placodal region (at the level of the presumptive nasal and lens placodes) or adjacent to the neural plate in the trunk (posterior to the pre-placodal region). Embryos were examined after 8 hours with a pan-Distalless antibody (pan-Dll) or an Eya2 probe, together with QCPN antibody to label quail tissue. Grafts adjacent to the anterior neural plate express both markers, but grafts adjacent to trunk level neural plate - which lie posterior to the pre-placodal region - do not. (B) Trunk ectoderm from young (0-2 ss) or old (7-9 ss) quail embryos was grafted into the pre-placodal region of 0-4 ss chick hosts. Embryos were examined after 8 hours with a pan-Distalless antibody (pan-Dll) or an Eya2 probe, together with QCPN antibody to label quail tissue. Young trunk ectoderm expresses both markers, but older ectoderm does not.

View larger version (61K): [in a new window]   Fig. 7. Upregulation of pre-placodal markers correlates with Fgf responsiveness. (A) Anterior epiblast was isolated from HH stage 3+-4 quail embryos and grafted into chick hosts at 0-4 ss. Grafts were placed either adjacent to the neural plate at the level of the presumptive trigeminal placode, or lateral to the presumptive trigeminal placode. Grafts were removed after 2 or 8 hours and cultured for a further 12 hours in the presence or absence of 50 ng/ml Fgf2. Control pieces of epiblast were isolated and cultured in Fgf2 for 20 hours without first being grafted into chick hosts. (B) Pax2 expression in cultured explants of transplanted quail epiblast, detected by QCPN expression. Pax2 is induced only by Fgf2 in explants grafted into the pre-placodal region for 8 hours. Two representative explants are shown for this condition, separated by broken lines. (C) Epiblast does not express Eya2 or label with pan-Dll antibodies after 20 hours of culture in the presence or absence of Fgf2.