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Morphoregulatory signaling systems: functional analysis of their impact on gene regulation during normal and altered embryonic development

We use a systems biology approach that combines mouse molecular genetics, transcriptome analysis and biochemistry with mathematical simulations to gain insight into the signalling and transcriptional networks that orchestrate vertebrate limb bud organogenesis. We study how progenitor cells are selected to act as embryonic organizers and how these control growth and patterning of limb buds. One of our main topics is to investigate how limb bud mesenchymal progenitors integrate various signaling inputs into a transcriptional response that regulates their survival, fates, proliferation and differentiation potential. For example, we have shown that the transcriptional regulator GLI3 initially regulates establishment of Sonic Hedgehog (SHH) signaling by the organizer in the posterior limb bud mesenchyme. GLI3 interacts with HAND2 as part of the transcription regulatory network that initiates establishment of an anterior and a posterior compartment prior to the onset of SHH signaling.

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As limb bud development progresses, GLI3 acts as a gatekeeper that regulates the exit of proliferating cells toward chondrogenic differentiation (Fig. 1). In particular, GLI3 directly regulates the cell-cycle and Expression of the BMP Antagonist Gremlin1 in limb buds. Our extensive genetic analysis has established that GREM1 is a key node within the self-regulatory signaling system that controls limb bud outgrowth and patterning. The highly dynamic Grem1 Expression is regulated by a large genomic landscape that integrates inputs from at least three different signaling pathways (BMP, SHH, FGF). Using 4C Chromatin conformation capture in combination with ChIP-sequencing, we are analysing how this integration works and how its self-regulatory feedback features are controlled. While this feedback system keeps BMP activity low during limb bud outgrowth and patterning, we have shown that high BMP activity is required during initiation for setting up the ectodermal signaling centre and then again at late stages to initiate chondrogenic differentiation.

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Other fascinating aspects of our research are briefly summarized below:

Evolutionary diversification of limbs in mammals: alterations in these signaling interactions not only cause congenital limb malformations, but also underlie evolutionary diversification of tetrapod limbs. We have recently been able to show that the loss of antero-posterior polarity and digits in bovine and artiodactyl limbs is due to degeneration of the cis-regulatory region that regulates the Expression of the Ptch1 receptor in response to SHH signaling. This loss of transcriptional sensitivity to SHH underlies the evolutionary adaptation of artiodactyl limbs, which diverged from other mammalians ~60 mio years ago.

Relevance to engineering of cartilage and bone from mesenchymal stem cells: the molecular networks controlling the dynamic modulation of TGFβ/BMP activity during limb bud development are highly relevant to the so-called developmental engineering of cartilage and bone from mesenchymal stem cells (MSCs). We are functionally analysing the molecular similarities and differences of normal chondrogenic differentiation and endochondral bone formation in embryonic limbs in comparison to induced differentiation of mouse MSCs.

The role of developmental modulators of SHH and BMP signaling in cancer: our genetic analysis shows that aberrant feedback signalling underlies malignant progression of medulloblastomas, which are the most common and deadly juvenile brain tumours in humans. In particular,we have shown that genetic reduction of SerpinE2, an extra-cellular modulator of the Hedgehog pathway interferes with progression of pre-neoplastic lesions to medulloblastomas in the Ptch1 heterozygous mouse model. Recently, we have obtained evidence that inactivation of Grem1 also lowers the incidence of medulloblastomas in Ptch1 mice.

In summary, we combine systematic genetic and cell-biochemical analysis with in silico simulations to gain insights into the complexity of the signalling networks that orchestrate normal organ and tissue development and aberrant malignant progression of medulloblastomas.