Identification of genes downstream of the Shh signalling in the developing chick wing and syn-expressed with Hoxd13 using microarray and 3D computational analysis

Abstract

Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid³ wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid³ wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid³ mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation.

Direct functional consequences of ZRS enhancer mutation combine with secondary long range SHH signalling effects to cause preaxial polydactyly

“…This suggested the creation of 19 potential transcription factor binding sites, of which 13 contained homeodomains (Table S1; Genomatix; Cartharius et al, 2005). While analysis of published and publically available gene expression patterns (GEISHA, Bell et al, 2004; eChickAtlas, Wong et al, 2013) with Affymetrix microarray expression analysis Wt and talpid 3 chicken limbs (Bangs et al, 2010) determined that most MatInspector candidates did not fulfil the candidate gene criteria (Table S1), HOXA13 , however, had appropriate spatiotemporal expression in Wt limbs. We therefore compared HOXA13 with HOXD13 in Slk/Wt , Wt/Wt embryos to confirm that it fulfilled our candidate criteria, as well as in talpid 3 embryos to confirm its responsiveness to SHH signalling.…”

A strategy to discover new organizers identifies a putative heart organizer

“…The following tissues were collected (three separate samples of each) and RNA extracted by standard methods: Hensen's Node at HH3 + /4 (HH4 HN) and HH5/6 (HH6 HN), posterior primitive streak at HH3 + /4 (HH4 PS), HH10/11 notochord and adjacent ventral neural tube/floor-plate (VNT) and the corresponding dorsal neural tube (DNT), the posterior third of the limb at HH20/21 (HH20 PL) and HH24 (HH24 PL) and the anterior third of the limb at HH20/21 (HH20 AL) and HH24 (HH24 AL)64.…”

Comparative Analysis of 3D Expression Patterns of Transcription Factor Genes and Digit Fate Maps in the Developing Chick Wing

“…For the GFP grafts, 3 replicates were mapped for each digit, where possible and a median again derived. We then divided each reference wing bud into spatial domains of 5×5×5 voxels as previously described [26], [37] with 945 spatial domains at stage 21, 2072 at stage 24 and 4444 at stage 27) and calculated mean signal intensity of expression of all the genes, including the GFP gene in each spatial domain. These data were then automatically tabulated in a tab delimited file to generate a matrix of gene expression patterns across all spatial domains.…”

“…Expression of these genes in both chick wings and mouse limb buds is regulated by the Shh/Bmp signalling cascade [22], although Tbx2 and Tbx3 may also be upstream of Shh [23], [24]. Recent analyses of transcriptional profiles of anterior versus posterior regions of both mouse limb and chick wing buds identified both Bmp2 and Sall1 as “posterior” genes [25], [26] and there are Gli binding sites upstream of these mouse genes [25].…”