The control of developmental plasticity between the external genitalia and the hindlimbs

During our study of mammalian embryogenesis, we uncovered a remarkable phenotype: fetuses with six legs and no external genitalia. Further investigation revealed that the additional limbs originated from the primordium typically responsible for genital development, demonstrating unexpected developmental plasticity and challenging conventional views on limb formation. Building on this discovery, our research aims to unravel how a single tissue can differentiate into either hindlimb or genital structures and explore its evolutionary implications.

Our findings also indicate that Tgfbr1 governs the genital primordium’s fate by reshaping chromatin accessibility, establishing distinct regulatory elements that other morphogenetic factors utilize to drive either genital or hindlimb development. We are now investigating the molecular mechanisms through which Tgfbr1 modulates the functional genomic architecture of these developmental pathways.

By integrating chromatin accessibility data with scRNA-seq analyses, followed by molecular and genetic validation, we aim to define the key gene regulatory networks that guide this tissue toward either a hindlimb or genital fate. The “six-legged” mouse model provides an exceptional opportunity to dissect these mechanisms and advance our understanding of developmental plasticity.

The control of Hox gene expression by Tgfbr1 signaling

Hox genes play a crucial role in controlling patterning processes along the anterior-posterior body axis in all bilaterian animals, including vertebrate limb buds. They form a large gene family organized into chromosomal clusters, with their number and composition varying across animal clades. Mammals possess 39 Hox genes, divided into four clusters (A to D) and 13 paralog groups—sets of genes with high sequence similarity that occupy equivalent positions in different clusters.

The linear arrangement of Hox genes within a cluster (from paralog 1 to paralog 13, in a 3’ to 5’ sequence) mirrors their expression pattern during development: 3’ genes are activated earlier and in more anterior regions, while 5’ genes are expressed later and more posteriorly. Despite extensive research, the stepwise activation mechanisms of Hox genes remain incompletely understood.

Our preliminary data suggest that Tgfbr1 may play a global role in activating 5’ Hox genes in both the main body axis and limb buds. However, the precise regulatory mechanisms remain unclear. To address this, we are employing gain- and loss-of-function experiments in mouse embryos, coupled with cutting-edge genomic approaches and functional analysis of regulatory elements, to elucidate how Tgfbr1 influences the activation of 5’ Hox genes in these developmental contexts.

– 2022-2025: 2022.01629.PTDC (from FCT, Portugal) (DOI: 10.54499/2022.01629.PTDC): “Coordinated development of the legs and external genitalia in vertebrates”.

– 2025-2026: 2023.12793.PEx (from FCT, Portugal) “Exploring the role of Tgfbr1 in the control of Hox genes expression in vertebrate limbs”.

Lozovska, A., Casaca, A., Nóvoa, A., Kuo, Y.-Y., Jurberg, A. D., Martins, G. G., Hadjantonakis, A.-K. & Mallo, M. (2024). Tgfbr1 regulates lateral plate mesoderm and endoderm reorganization during the trunk to tail transition.eLife 13, RP94290. doi: 10.7554/eLife.94290.

Lozovska, A., Korovesi, A. G., Dias, A., Lopes, A., Fowler, D. A., Martins, G. G., Nóvoa, A. & Mallo, M. (2024). Tgfbr1 controls developmental plasticity between the hindlimb and external genitalia by remodeling their regulatory landscape. Nature Communications 15, 2509. doi: 10.1038/s41467-024-46870-z.

Duarte, P., Brattig Correia, R. Nóvoa, A. & Mallo, M. (2023). Regulatory changes associated with the head to trunk developmental transition. BMC Biology 21, 170. doi: 10.1186/s12915-023-01675-2.

Dias, A., Lozovska, A., Wymeersch, F.J., Nóvoa, A., Binagui-Casas, A., Sobral, D., Martins, G.G., Wilson, V. & Mallo, M. (2020). A Tgfbr1/Snai1-dependent developmental module at the core of vertebrate axial elongation. eLife 9, e56615. Doi: 10.7554/eLife.56615

Aires, R., de Lemos, L., Nóvoa, A., Jurberg, A. D., Mascrez, B., Duboule, D. & Mallo, M. (2019). Tail bud progenitor activity relies on a network comprising Gdf11, Lin28 and Hox13 genes. Dev. Cell 48, 383-395. Doi: 10.1016/j.devcel.2018.12.004

Mallo, M. (2018). Reassessing the role of Hox genes during vertebrate development and evolution. Trends in Genetics 34, 209-217. Doi: 10.1016/j.tig.2017.11.007

Aires, R., Jurberg, A. D., Leal, F., Nóvoa, A., Cohn, M. J. & Mallo, M. (2016). Oct4 is a key regulator of vertebrate trunk length diversity. Dev. Cell 38, 262-274. Doi: 10.1016/j.devcel.2016.06.021

Jurberg, A. D., Aires, R., Varela-Lasheras, I., Nóvoa, A. & Mallo, M. (2013). Switching axial progenitors from producing trunk to tail tissues in vertebrate embryos. Dev. Cell 25, 451-462. Doi: 10.1016/j.devcel.2013.05.009

Guerreiro, I., Nunes, A., Woltering, J., Casaca, A., Nóvoa, A., Vinagre^, T., Hunter, M. E., Duboule, D. & Mallo, M. (2013). Role of a polymorphism in a Hox/Pax-responsive enhancer in the evolution of the vertebrate spine. Proc. Natl. Acad. Sci. USA 110, 10682-10686. Doi: 10.1073/pnas.1300592110

Mallo, M., Wellik, D. M. & Deschamps, J. (2010). Hox genes and regional patterning of the vertebrate body plan. Dev. Biol. 344, 7-15. Doi: 10.1016/j.ydbio.2010.04.024

A complete list of publications can be found here. 

2005, Pfizer Prize for Basic Research

2010, Pfizer Prize for Basic Research