A Switch from Low to High Shh Activity Regulates Establishment of Limb Progenitors and Signaling Centers

Zhulyn, Olena; Li, Danyi; Deimling, Steven; Vakili, N Alizadeh; Mo, Rong; Puviindran, Vijitha; Chen, Miao-Hsueh; Chuang, Pao-Tien; Hopyan, Sevan; Hui, Chi‐chung

doi:10.1016/j.devcel.2014.03.002

Cited by 45 publications

(93 citation statements)

References 34 publications

(66 reference statements)

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“…In particular, simultaneous inactivation of Irx3/5 and Gli3 caused failure to establish a Shh-expressing ZPA, and caused trace Fgf8 expression in the AER. However, this phenotype was hindlimb specific and forelimbs developed polydactyly, similar to Gli3 KO limbs (11). In significant contrast to this report, we found that Sall4-Gli3 cooperation regulates limb signaling center development in both fore-and hindlimbs.…”

Section: Discussioncontrasting

confidence: 56%

“…Gli3 functions for specifying anterior-posterior polarity in the nascent limb buds (9), and Gli3 null embryos develop polydactyly without polarity (10). Furthermore, simultaneous inactivation of Gli3 and Irx3/5 caused failure to express Shh and significantly reduced levels of Fgf8, which resulted in severe limb truncation (11). This result suggests that cooperation of anterior genes is upstream of establishing limb signaling centers, and therefore expansion of SHH-dependent posterior progenitors.…”

mentioning

confidence: 66%

“…2F). We also monitored Fgf8 expression, given a recent genetic study, involving Irx3/5 and Gli3, indicated that cooperation of anterior genes is necessary for establishing limb signaling centers, such as Shh-expressing ZPA and Fgf8-expressing AER (11).…”

Section: Sall4mentioning

confidence: 99%

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Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements

Akiyama

Kawakami

Wong

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore-and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.Sall4 | Gli3 | limb progenitors | appendicular skeletal elements | Plzf-Hox H ow progenitor cells are spatially and temporarily organized to construct an organ is a central question in developmental biology. Limb skeletal elements develop from limb progenitors, which arise from the lateral plate mesoderm (LPM) that is originated from epithelial somatopleure (1). Limb progenitors initially form two paired protrusions, fore-and hindlimb buds, whose initiation occurs around embryonic day (E) 9.0 and E9.5, respectively, in mouse embryos. In the following steps, limb signaling centers, known as the zone of polarizing activity (ZPA) and apical ectodermal ridge (AER), are established. SHH (Sonic hedgehog) from the ZPA and FGF8 from the AER are major signal molecules that regulate proliferative expansion and patterning of early limb progenitor cells (reviewed in ref. 2). These processes lead to development of functional limbs with each skeletal element adopting a unique shape at a distinct location.Several studies suggest that limb progenitors consist of two distinct pools, an anterior progenitor pool and a posterior progenitor pool. The posterior progenitor pool consists of cells that once expressed Shh and cells that received paracrine effects of SHH, which contribute to digit 2 (d2), d3, d4, and d5 and the posterior zeugopod (ulna, fibula) (3-6). Contrary to this, the anterior ...

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Section: Discussioncontrasting

confidence: 56%

mentioning

confidence: 66%

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Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements

Akiyama

Kawakami

Wong

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…3G, H, J, K). These phenotypes were consistent with compromised Gli3 REP function [31–33]. To determine whether digit defects in Sufu T396I/T396I are due to impaired Gli3 REP activity, we forced expression of a constitutive Gli3 REP allele ( Gli3 ∆699 ), which produces a truncated protein due to a premature termination codon [34], in the Sufu T396I/T396I background.…”

Section: Resultsmentioning

confidence: 99%

T396I Mutation of Mouse Sufu Reduces the Stability and Activity of Gli3 Repressor

Makino

Zhulyn

et al. 2015

PLoS ONE

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Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.

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“…Sufu knock-out mice display early embryonic lethality at E9.5, with severe cranio-facial and neural closure defects related to constitutive SHH pathway activation, 31 similar to that observed in Ptch1 mutants. 32 Limb-specific conditional deletion of Sufu results in polydactyly due to a block in Gli3R processing, 33,34 whereas mouse embryos with conditional knock-out of Sufu in the mid-hindbrain show altered morphology of the brainstem and cerebellum and delayed differentiation and abnormal migration of most cerebellar cell types. 35 Here, we show that germline hypomorphic variants of SUFU cause deregulation of the SHH pathway, resulting in recessive stereotypical developmental defects of the CNS and limbs which share peculiar features with both SHH-related disorders and with ciliopathies such as JS.…”

Section: Introductionmentioning

confidence: 99%

Hypomorphic Recessive Variants in SUFU Impair the Sonic Hedgehog Pathway and Cause Joubert Syndrome with Cranio-facial and Skeletal Defects

Mori

Romani²,

D’Arrigo

et al. 2017

The American Journal of Human Genetics

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The Sonic Hedgehog (SHH) pathway is a key signaling pathway orchestrating embryonic development, mainly of the CNS and limbs. In vertebrates, SHH signaling is mediated by the primary cilium, and genetic defects affecting either SHH pathway members or ciliary proteins cause a spectrum of developmental disorders. SUFU is the main negative regulator of the SHH pathway and is essential during development. Indeed, Sufu knock-out is lethal in mice, and recessive pathogenic variants of this gene have never been reported in humans. Through whole-exome sequencing in subjects with Joubert syndrome, we identified four children from two unrelated families carrying homozygous missense variants in SUFU. The children presented congenital ataxia and cerebellar vermis hypoplasia with elongated superior cerebellar peduncles (mild ''molar tooth sign''), typical cranio-facial dysmorphisms (hypertelorism, depressed nasal bridge, frontal bossing), and postaxial polydactyly. Two siblings also showed polymicrogyria. Molecular dynamics simulation predicted random movements of the mutated residues, with loss of the native enveloping movement of the binding site around its ligand GLI3. Functional studies on cellular models and fibroblasts showed that both variants significantly reduced SUFU stability and its capacity to bind GLI3 and promote its cleavage into the repressor form GLI3R. In turn, this impaired SUFU-mediated repression of the SHH pathway, as shown by altered expression levels of several target genes. We demonstrate that germline hypomorphic variants of SUFU cause deregulation of SHH signaling, resulting in recessive developmental defects of the CNS and limbs which share features with both SHH-related disorders and ciliopathies.

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A Switch from Low to High Shh Activity Regulates Establishment of Limb Progenitors and Signaling Centers

Cited by 45 publications

References 34 publications

Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements

Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements

T396I Mutation of Mouse Sufu Reduces the Stability and Activity of Gli3 Repressor

Hypomorphic Recessive Variants in SUFU Impair the Sonic Hedgehog Pathway and Cause Joubert Syndrome with Cranio-facial and Skeletal Defects

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