Congenital limb malformations, caused by abnormal limb development, occur in one in 1,000 live human births. Therefore, understanding the mechanisms of limb development is relevant to biology and medicine. Limb development starts with the specification of a discrete region of the lateral plate mesoderm into limb progenitors, which gives rise to the limb bud. In the last several decades, the research field intensely focused on understanding the mechanisms by which signaling centers in limb buds regulate patterning of limb buds, leading to formation of limb skeletons. However, we have limited knowledge about how limb progenitors are specified and what mechanisms regulate their initial differentiation before the establishment of limb bud signaling centers; yet, these processes are essential for correct limb development. Studies in the last decades showed that distinct mechanisms operate on lateral plate mesoderm and limb progenitors prior to establishing limb bud signaling centers. For example, we found that deletion of Sall4, encoding a zinc finger transcription factor, approximately two days before the onset of limb development, resulted in severe defects specifically in hindlimbs, while deletion at later stages had no or subtle effect. In our preliminary studies, we found that simultaneous inactivation of Sall4, Irx3 and Irx5 (Irx3/5) caused the absence of hindlimbs with the loss of expression of hindlimb progenitor-specific genes, such as Isl1. This result indicates that combined function of Sall4 and Irx3/5 specifies lateral plate mesoderm into hindlimb progenitors.
In Aim 1, our goal is to elucidate the molecular mechanisms of hindlimb progenitor specification. We will determine whether SALL4 and IRX3/5 redundantly and directly regulate Isl1 through its enhancer. We will determine genes that act downstream of Sall4 and Irx3/5 to specify hindlimb progenitors by genomic experiments. We will determine their functions in specifying hindlimb progenitors by genetic knockout approaches. We also obtained data, strongly suggesting that Sall4 knockout causes increased glycolysis in limb progenitors, when endogenous glycolysis is transitioning from high to low activity. Recent studies provided evidence that, beyond supplying energy, glycolysis mediates fibroblast growth factor signaling in the tail bud and regulates body elongation. Fibroblast growth factor signaling is one of earliest signaling that regulates limb progenitor differentiation.
In Aim 2, we will test an intriguing hypothesis that Sall4-dependent repression of glycolysis regulates differentiation of limb progenitors. We will construct metabolomes of wild type and Sall4 mutant limb progenitors to understand metabolic status and the changes by loss of Sall4. We will test the role of glycolysis by reducing glycolysis in Sall4 mutant embryos and determine their differentiation, as well as increasing glycolysis in embryos without mutations. This proposal will generate important basic information on the specification and differentiation of limb progenitors, which are fundamental initial processes of limb development.

Public Health Relevance

/RELEVANCE In order to generate specific skeletal progenitor cells that may be used for treatments of skeletal disorders by regenerative medicine approaches, we must understand how progenitors arise and begin to differentiate during normal development. The proposed studies are designed to gain mechanistic insights into how limb progenitors are generated and differentiate. The results will enhance our understanding of the biology of limb progenitors, which would provide a foundation for developing methods to generate limb skeletal precursors for regenerative medicine.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR064195-08
Application #
10005889
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Kirilusha, Anthony G
Project Start
2013-04-01
Project End
2023-08-31
Budget Start
2020-09-01
Budget End
2021-08-31
Support Year
8
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Genetics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Kawakami, Hiroko; Johnson, Austin; Fujita, Yu et al. (2018) Characterization of cis-regulatory elements for Fgf10 expression in the chick embryo. Dev Dyn 247:1253-1263
Tahara, Naoyuki; Akiyama, Ryutaro; Theisen, Joshua W M et al. (2018) Gata6 restricts Isl1 to the posterior of nascent hindlimb buds through Isl1 cis-regulatory modules. Dev Biol 434:74-83
Tao, Hirotaka; Kawakami, Yasuhiko; Hui, Chi-Chung et al. (2017) The two domain hypothesis of limb prepattern and its relevance to congenital limb anomalies. Wiley Interdiscip Rev Dev Biol 6:
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Yu, Xiaodan; Kawakami, Hiroko; Tahara, Naoyuki et al. (2017) Expression of Noggin and Gremlin1 and its implications in fine-tuning BMP activities in mouse cartilage tissues. J Orthop Res 35:1671-1682
Yokoyama, Shigetoshi; Furukawa, Soichi; Kitada, Shoya et al. (2017) Analysis of transcription factors expressed at the anterior mouse limb bud. PLoS One 12:e0175673
Baik, June; Magli, Alessandro; Tahara, Naoyuki et al. (2016) Endoglin integrates BMP and Wnt signalling to induce haematopoiesis through JDP2. Nat Commun 7:13101
Hayashi, Shinichi; Akiyama, Ryutaro; Wong, Julia et al. (2016) Gata6-Dependent GLI3 Repressor Function is Essential in Anterior Limb Progenitor Cells for Proper Limb Development. PLoS Genet 12:e1006138
Tahara, Naoyuki; Brush, Michael; Kawakami, Yasuhiko (2016) Cell migration during heart regeneration in zebrafish. Dev Dyn 245:774-87

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