A reduction of primary cilia but not hedgehog signaling disrupts morphogenesis in testicular organoids

Abstract

Most mammalian cells possess a single, non-motile primary cilium that plays an important role in mediating cellular signaling pathways, such as Hedgehog (Hh) signaling. Primary cilia are present on testicular somatic cells and demonstrate a temporal expression during development; however, their role in testicular morphogenesis is not well characterized. To investigate the role of primary cilia and Hh signaling in Sertoli cells on morphogenesis, we inhibited assembly of primary cilia through CRISPR Cas9-mediated gene editing of ODF2, a structural component of primary cilia and siRNA-mediated gene silencing of IFT88, a functional component of the intraflagellar transport system. Knockdown of ODF2 and IFT88 resulted in a 50% reduction in the number of cells with primary cilia and significant shortening of the remaining cilia. The expression of GLI1, a downstream target of Hh signaling, was significantly reduced when IFT88 but not ODF2, was downregulated. When morphogenesis was examined using tubule formation in vitro and a novel testicular organoid system, loss of cilia after knockdown of both targets affected cellular assembly and organization. While the Hh pathway was found to be active during morphogenesis in vitro, addition of the Hh antagonist cyclopamine did not affect morphogenesis in either in vitro system. These results indicate that primary cilia are important for morphogenesis in vitro but Hh signaling is not the cilia-mediated pathway responsible for orchestrating morphogenic organization.

Keywords: Hedgehog signaling; Morphogenesis; Organoids; Primary cilia; Sertoli cells.

Fig. 1

Editing of ODF2 and silencing of IFT88 reduces the number and length of primary cilia. a, d Relative expression of target mRNA. In a, n = 3. In d, n = 5. Bars indicate minimum and maximum RQ. b, e Percent of cells possessing primary cilia. In b, control n = 6, ODF2e n = 4. In e, control n = 7, IFT88si n = 6. Bars indicate ± SEM. c, f Length of primary cilia. In c, control n = 493, ODF2e n = 483. In f, control n = 371, IFT88si n = 400 cells counted. Bars indicate minimum and maximum ciliary lengths measured; center line is median length

Fig. 2

Knockdown of IFT88 but not ODF2 affects cellular proliferation. a, b, d, e EdU-positive cells in control and treated populations. Scale bars 100 μm. c, f Quantification of EdU-positive cells in control and treated populations (n = 3). Bars indicate ± SEM

Fig. 3

Effect of targeting ODF2 and IFT88 on GLI1 expression upon treatment with SAG. a Control and ODF2e cells, n = 3. b Control and IFT88si cells, n = 4. Bars indicate minimum and maximum RQ

Fig. 4

Reduction of primary cilia impairs formation of tubules in vitro. a, b Tubules made from control and ODF2e cells. Scale bars 5 mm. c, d Higher magnification of tubules in a and b. Scale bars 500 μm. e, f Tubules formed from control and IFT88si cells. Scale bars 500 μm

Fig. 5

Reduction of primary cilia impairs formation of organoids in vitro. a, b Organoids formed from control and ODF2e cells. Scale bars 50 μm. c, d Organoids formed from control and IFT88si cells. Scale bars 50 μm. e, f Basement membrane deposition and cellular organization of organoids from control and IFT88si cells. Scale bars 40 μm

Fig. 6

Hedgehog signaling is active but not essential for tubule morphogenesis in vitro. a GLI1 expression (blue line) over the course of 6 days in in vitro tubules. Day 0, n = 7; day 1, n = 5; days 2–6, n = 4. GLI1 expression upon treatment with cyclopamine (red line). n = 3. Bars indicate minimum and maximum RQ. b Tubule formation at 1 and 3 days. Scale bars 500 μm. c Tubule formation at 1 and 3 days upon treatment with cyclopamine. Scale bars 500 μm

Fig. 7

Hedgehog signaling in organoids was not essential for morphogenesis. a GLI1 expression over 4 days (blue line) in organoids. n = 3. GLI1 expression upon treatment with cyclopamine (red line). n = 3. Bars indicate minimum and maximum RQ. b, c Appearance of organoids treated with DMSO vehicle control or cyclopamine. Scale bars 50 μm. d, e Basement membrane deposition and cellular organization of control organoid and organoid formed from cells treated with cyclopamine. Scale bars 25 μm