# Role of intraflagellar transport protein IFT140 in the formation and function of motile cilia in mammals

Yi Tian Yap, Jiehong Pan, Jian Xu, Shuo Yuan, Changmin Niu, Cheng Zheng, Wei Li, Ting Zhou, Tao Li, Yong Zhang, et al.

► To cite this version:

Yi Tian Yap, Jiehong Pan, Jian Xu, Shuo Yuan, Changmin Niu, et al., Role of intraflagellar transport protein IFT140 in the formation and function of motile cilia in mammals. Cellular and Molecular Life Sciences, 2025, 82 (1), pp.198-216. 10.1007/s00018-025-05710-z, hal-05393866

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# Role of intraflagellar transport protein IFT140 in the formation and function of motile cilia in mammals

Yi Tian Yap¹ · Jiehong Pan² · Jian Xu² · Shuo Yuan¹,³ · Changmin Niu¹,⁴ · Cheng Zheng¹,³ · Wei Li¹ · Ting Zhou¹,³ · Tao Li¹,⁵ · Yong Zhang² · Michael J. Holtzman² · Gregory J. Pazour⁶ · Rex A. Hess⁷ · Christopher V. Kelly⁸ · Aminata Touré⁹ · Steven L. Brody² · Zhibing Zhang¹,¹⁰

Received: 21 September 2024 / Revised: 5 April 2025 / Accepted: 10 April 2025
© The Author(s) 2025

## Abstract

Cilia are microtubular structures extending from the surface of most mammalian cells. They can be categorized as motile cilia and primary sensory cilia. Both types possess intraflagellar transport (IFT) machinery, composed of unique protein complexes that travel along the microtubules to deliver proteins for ciliary and flagellar assembly, disassembly, and homeostasis. Although the role of IFT in primary cilia formation has been well studied, little is known about its role in mammalian motile cilia assembly. We generated conditional knockout mice by breeding floxed *Ift140* mice with the *FOXJ1*-Cre transgenic mouse line to specifically delete *Ift140* from cells that assemble motile cilia. Mice with *Ift140* deficiency did not have laterality defects or gross; however most died prior to sexual maturity. Those mutants that survived to adulthood were completely infertile. Males demonstrated abnormal spermatogenesis associated with reduced sperm count and motility, together with short length flagella, and abnormal morphology. Cilia length was diminished in the epithelial cells of the efferent ductules and airways. Cilia from cultured tracheal epithelial cells were also short and had reduced beat frequency (CBF). Ultrastructural studies revealed the presence of inner and outer dynein arms, but an abnormal central apparatus, and the accumulation of particles within the cilia. Overall, the short length and abnormal localization of ciliary proteins in *Ift140* conditional mutants resulted in inadequate cilia function despite proper localization of the dynein motor complexes. We propose a key role of *Ift140* for motile cilia assembly in certain tissues and suggest that genetic alterations of *IFT140* could be associated with motile ciliopathies.

**Keywords** Cilia · Fertility · Intraflagellar transport · Sperm

## Introduction

Cilia are microtubular structures present at the surface of most mammalian cells [1]. The two major subtypes of cilia are primary cilia and motile cilia which differ both structurally and functionally. Primary cilia are cellular protrusions commonly found on the apical region of many cell types and predominantly serve sensory functions. They are comprised of 9 microtubule doublets, giving them the characteristic “9 + 0” structure. On the other hand, motile cilia are present in sperm and the multiciliated cells lining the brain ventricles,

the respiratory tract, the oviduct in females, and the efferent ducts in males. The typical structure of a motile cilium is comprised of 9 microtubule doublets and a pair of central microtubules, resulting in the “9 + 2” configuration. Essential characteristic structures present in the motile cilia are dynein arms that facilitate ciliary beat and movement [2].

Even though it was reported that ribosomes are also present in the motile cilia [3], it has been well established that the components of the cilia are produced within the cell body and then transported to the developing cilium. Intraflagellar transport (IFT) is the cargo transport system that facilitates the assembly and maintenance of both primary and motile cilia. The bidirectional IFT transport was discovered by Rosenbaum and colleagues using differential interference contrast microscopy to observe trafficking in paralyzed *Chlamydomonas* mutants [4]. Further studies showed that IFT is required for the assembly of all types

Yi Tian Yap, Jiehong Pan, Jian Xu and Shuo Yuan have contributed equally in this study.

Steven L. Brody, and Zhibing Zhang are the co-senior authors.

Extended author information available on the last page of the article
of cilia, including mammalian [5, 6]. Two multimeric IFT complexes, traveling in opposite directions, enable bidirectional particle transport along the axoneme. IFT complex A consists of IFT43, IFT121, IFT122, IFT139, IFT140, and IFT144 for retrograde transport. While IFT complex B comprises of 16 proteins subcategorized into IFT-B1 (IFT22, IFT25, IFT27, IFT46, IFT52, IFT56, IFT70, IFT74, IFT81, IFT88) and IFT-B2 (IFT20, IFT38, IFT54, IFT57, IFT80, and IFT172) for anterograde transport [7]. During anterograde transport, cargo proteins are conveyed from the cytoplasm to the tips of the cilia and sustain cilia growth and homeostasis. Subsequently, turnover products are transported from the tip back to the base of the cilia for recycling through retrograde transport [8, 9]. Dedicated motor proteins, kinesins and dyneins, mediate anterograde and retrograde IFT trafficking, respectively [10, 11]. IFT has been predominantly studied in primary cilia with recent focus on genetic variants. Defects in the IFT machinery are associated with many human syndromic ciliopathies, including Bardet-Biedl syndrome, Sensenbrenner syndrome, Mainzer-Saldino syndrome, and Jeune syndrome [12-16], as well as isolated retinal degeneration, polycystic kidney disease, and male infertility [17-20]. Primary ciliary dyskinesia (PCD) is the genetic disorder caused by motile cilia dysfunction, resulting in a range of clinical manifestations affecting the upper and lower respiratory tract, reproductive system, and left-right body asymmetry [21]. However, the precise role of IFT genes in motile cilia assembly and maintenance and related diseases such as PCD, including male infertility remains unclear. With the exception of *Ift74* [22], most patients with pathogenic *IFT* variants are not known to have respiratory defects. Studies in mice show that mutations in *Ift* genes like *Ift88* [23] significantly impact respiratory and other multiciliated cell types, suggesting similar phenotypes should be observed in the human population. However, perinatal lethality and other severe non-respiratory pathologies resulting from the same mutations in *IFT* genes could obscure respiratory dysfunction. In addition, it is possible that pathogenic variants in humans are hypomorphic and may not be strong enough to result in significant respiratory complications. Thus, the importance of *Ift* in multiciliated cells is relatively unexplored.

We have previously studied several mouse models with conditional deficiency in selective *Ift* genes induced in adult mice during spermiogenesis, with a focus on the IFT-B complex components. We reported that disruption of *Ift20*, *Ift25*, and *Ift27* in male germ cells resulted in infertility [24-26]. Likewise, IFT81 male mice were infertile associated with reduced IFT-B components in testes, and others, including IFT20, IFT25, IFT27, IFT57, IFT74, and IFT88, but there was no change in the IFT-A complex protein IFT140 [27]. We reported that conditional deletion of IFT140 in

spermatocytes and spermatids resulted in infertility [28], implicating a requirement for IFT-A component in mammalian motile ciliogenesis in sperm. The requirement of specific IFT components in other motile cilia has not been widely studied. To address this, we deleted a floxed allele of *Ift140* in cells with motile cilia using *FOXJ1-Cre*.

Forkhead box J1 (Foxj1) serves as the master regulator of motile ciliogenesis. This transcription factor is almost exclusively expressed in ciliated cells present in the respiratory tract, choroid plexus, ependyma, oviduct, efferent ducts, and embryonic nodal regions [29-32]. Inhibition of Foxj1 results in compromised assembly of motile cilia in zebrafish and Xenopus [33]. In mice during lung development, the expression of *Foxj1* was first detected at embryonic day 14.5, just prior to the appearance of cilia [29]. Mice mutants lacking Foxj1 expression demonstrate the absence of cilia in airways, left-right axis defects, hydrocephalus, and infertility [30, 34]. A similar phenotype including left-right defects, hydrocephalus, and perinatal mortality is observed in mice deficient in genes regulated by Foxj1 that are commonly deficient in PCD, such as the murine orthologues of DNAH5, DNAI1, DNAAF5, CCDC39, and CCDC40 [35-40]. As such, the established mouse line containing the *FOXJ1-Cre* transgene provides a tool for investigating the role of genes in the mammalian motile cilia [41].

To expand our knowledge regarding the role of IFT in motile cilia, we bred floxed *Ift140* mice with *FOXJ1-Cre* mice. This breeding strategy yielded mice lacking the specific IFT140 protein exclusively in motile cilia. *Ift140 flox/flox*; *FOXJ1-Cre* (*Ift140* cKO) mice survived to adulthood, but were infertile, with the males exhibiting defects in spermatogenesis and altered motile cilia in the efferent ductules. The remaining *Ift140* cKO mice displayed delayed growth, and death at less than three weeks of age. Cilia from tracheal epithelial cells (mTEC) cultured from the *Ift140* cKO mice were less numerous and exhibited decreased ciliary beat frequency (CBF). Electron-dense particles were observed in the cilia of *Ift140* cKO mTEC. Sperm associated gene 16L (SPAG16) and sperm associated gene 17 (SPAG17), key central pair components, were mislocalized in the epithelial cells. These findings strongly support the involvement of IFT140 in the formation of mammalian motile cilia.

## Results

### Generation of a *FOXJ1-Cre* mediated *Ift* mutant mouse model

To investigate the role of IFT140 in motile cilia, we bred floxed *Ift140* with *FOXJ1-Cre* mice to generate conditional knockout mice, where the *Ift140* gene was specifically disrupted in the motile cells, all in a C57BL/6 background

<figure><img src="image_2.png" /></figure>
(Supplemental Fig. 1). Genotyping confirmed the acquisi-
tion of homozygous mutant mice (Supplemental Table 1).
*Ift140*<sup>flox/+</sup>; littermates that were *FOXJ1-Cre*<sup>±</sup> or *Ift140*<sup>flox/flox</sup>
mice negative for *FOXJ1-Cre* were used as controls.

**Ift140 cKO mice that survived to adulthood exhibited infertility had spermiogenesis defects**

The first generation of *Ift140* cKO mice (*Ift140*<sup>flox/flox</sup;
*FOXJ1-Cre<sup>±</sup>*) did not exhibit gross abnormalities such as
hydrocephalus, and they had normal left-right asymmetry.
However, none of the homozygous males and females *Ift140*
cKO mice were able to sire pups over a period of more
than two months of breeding (Table 1). Spermatogenesis
was evaluated in the *Ift140* cKO adult males. Histological
examination of the testes revealed an abnormal spermatato-
genesis process in the *Ift140* cKO mice. In control mice,
immature germ cells were aligned along the basal region
of the tubules, while mature sperm were found within the
tubule lumen (Fig. 1A). Conversely, in the *Ift140* cKO mice,
the seminiferous tubules displayed an enlarged lumen and
were lined with a thinner layer of cells (Fig. 1A). Numerous
mature sperm were concentrated in the cauda epididymis of
control mice (Fig. 1B). However, in the *Ift140* cKO mice,
the cauda epididymal lumen contained fewer sperm, along
with sloughed round spermatids and cell debris (Fig. 1B).
Consistently, sperm count was significantly reduced in the
*Ift140* cKO mice (Fig. 1C). Further examination of sperm
morphology at high magnification using light microscopy
revealed that sperm from the control mice exhibited normal
morphology whereas most sperm from the *Ift140* cKO mice
were short and had bent tails with altered head morphol-
ogy (Fig. 1D). Additionally, while sperm from the control
mice demonstrated normal motility (Supplemental movie 1,
Fig. 1E), the few sperm from the *Ift140* cKO mice exhibited
reduced motility (Supplemental movie 2, Fig. 1E).

In wild-type mice, motile cilia are present in the effer-
ent ductules and the beating of these cilia is known to be
responsible for stirring or creating luminal turbulence for
proper reabsorption of nearly 90% of the fluid [42]. It has
been reported that dysfunction or loss of motile cilia in the
fferent ductules disturbs the luminal homeostasis, caus-
ing sperm accumulation, occlusion of the efferent ducts,
and buildup of fluid within the testis, all of which result
in male infertility [43–45]. Consequently, the efferent duct-
ules were examined. A strong and continuous ciliary sig-
nal was observed in the epithelial cells along the lumen in
control mice (Fig. 1F, upper panel). However, in the *Ift140*
cKO mice, the ciliary signal was scattered along the lumen
(Fig. 1F, lower panel). Histology of the efferent ductules was
also examined. In contrast to the control mice, the *Ift140*
cKO mice exhibited a much larger lumen with sperm trapped
inside (Fig. 1G, upper panel). The few TEM cross-sectional

images of the axoneme of an *Ift140* cKO mouse suggested
a disrupted central pair (Fig. 1G, lower panel). The major
phenotype observed in male *Ift140* cKO mice was summa-
rized in Fig. 1H.

**Ift140 cKO mice exhibited growth retardation and early death after the first generation**

Although the first generation of *Ift140* cKO mice did not
exhibit gross abnormalities, starting from the second gen-
eration, all the homozygous mutant mice began to show
growth retardation and eventually died before reaching
three weeks of age. To closely monitor this process, the
body weight of *Ift140* cKO mice was measured weekly.
Upon birth, no differences were observed among the vari-
ous genotypes (Fig. 2A). However, by approximately one
week of age, the *Ift140* cKO mice exhibited smaller size,
and by two weeks of age, their size was dramatically less
than their control littermates (Fig. 2B). Both male and
female (Fig. 2Cb) *Ift140* cKO mice had significantly lower
body weights compared to controls at both weeks 1 and
2 of age (Fig. 2Ca, Cb). All *Ift140* cKO mice succumbed
before reaching three weeks of age. No instances of hydro-
cephalus (Fig. 2D) or situs inversus were observed in these
mice and cilia in the brain ventricles appeared unaffected
as indicated by immunostaining of ependymal cilia in con-
trol and *Ift140* cKO mice (Supplement Fig. 2). Histology
of major organs, including the kidney and liver, was exam-
ined in the control and *Ift140* cKO mice. No cystic changes
found in mice with primary cilia defects were observed in
any of these organs (Supplemental Fig. 3).

**Ift140 cKO mice had abnormal cilia formation in the airway epithelial cells**

Despite the survival of the first generation of *Ift140* cKO
mice to adulthood, all mutants were infertile, suggesting
ineffective function of motile cilia in the efferent ductules
and oviduct. The remaining homozygous mutant mice died
before reaching three weeks of age, posing a challenge for
analyzing all organs with cilia of these small mice. There-
fore, we analyzed only the trachea and lungs. Immunofluo-
rescence staining revealed a near continuous acetylated
tubulin signal in the epithelial cells lining the trachea, bron-
chi, and intrapulmonary airways of control mice (Fig. 3A,
upper panel). Conversely, the *Ift140* cKO mice exhibited
only scattered ciliary signals (Fig. 3A, lower panel). Histo-
logical examination of the trachea unveiled morphological
differences between control and *Ift140* cKO mice. In con-
trol mice, the lumen was lined with cilia projecting into the
lumen (Fig. 3B, upper panel). However, in *Ift140* cKO mice,
fewer cilia were observed in the epithelial cells, and these
cilia were shorter (Fig. 3B, lower panel; Fig. 3C). To further
A

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B

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C

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D

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E

Percentage of motile sperm.

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F

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G

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H

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assess the cilia observed in the trachea of mice, mTEC were cultured from both control and *Ift140* cKO mice. Analysis of ciliogenesis of cultured cells from control and cKO mice showed similar centriologenesis and basal body docking, but decreased cilia number in the cKO preparations (Fig. 4). Abundant multiciliated cells were observed in control mTEC (Supplemental Fig. 4, left three panels). In contrast, *Ift140* cKO mTEC had fewer multiciliated cells, often with sparse cilia (Supplemental Fig. 4, right two panels). Examination of cilia length assessed by immunofluorescence staining and TEM in mTEC cultures revealed that *Ift140* cKO had noticeably shorter cilia compared to control mTEC (Fig. 5).

Cilia from the *Ift140* cKO mTEC were also functionally abnormal, as evidenced by the reduced cilia beat frequency (CBF) (Fig. 6). Cilia beat frequency was analyzed in cells isolated by scraping cells from tracheal surface and in cultured mTEC. Compared to controls, ciliary beating frequency was significantly reduced in cells isolated from the *Ift140* cKO trachea (Fig. 6A, Supplemental Fig. 5, Supplemental movie 3) and in cultured mTEC (Fig. 6B, Supplemental movie 4).

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►Fig. 1 Sperm formation was affected in the surviving conditional *Ift140* cKO mice. A Representative testicular histology from a control mouse and three *Ift140* cKO mice. Notice that the lumen of the seminiferous tubules in *Ift140* cKO mice were highly dilated (arrow heads); a number of degenerated germ cells were sloughed in the lumen (arrows); B Representative epididymis histology from a control mouse and a *Ift140* cKO mouse. Notice that the lumen was filled with sperm in the control mouse (dashed arrows) while few sperm cells were detected in *Ift140* cKO mice. C Significantly reduced sperm count in *Ift140* cKO mice. Left: representative images at low magnification of epididymal sperm collected from a control mouse and three *Ift140* cKO mice. Notice that the sperm density was lower in the three cKO mice when the samples were prepared in the same dilution; right: statistical analysis of sperm count from three control mice and three *Ift140* cKO mice. *p* < 0.01. D Abnormal sperm morphology in *Ift140* cKO mice. a Representative high magnification sperm images from a control mouse and three *Ift140* cKO mice. Most sperm from *Ift140* cKO mice had bent tails (arrows); b Average length of sperm flagella of the control and *Ift140* cKO mice. 50 sperm with straight tails were randomly selected from four control and three *Ift140* cKO mice and the tail length were measured. *P* < 0.01. E Significantly reduced sperm motility in the *Ift140* cKO. a Percentage of motile sperm; b Sperm motility. *P* < 0.01. F Reduced cilia signal on the efferent ductules of the *Ift140* cKO mice compared to controls. Representative images of immunofluorescence staining conducted on efferent ductules closer to the testes side collected from a control mouse and a *Ift140* cKO mouse, using an anti-acetylated tubulin antibody. Strong cilia signal (green arrows) was visible in the control mouse. However, few cilia signals were detected in *Ift140* cKO mice. The lumen was dilated and more sperm (white arrows) were present in the efferent ductules of *Ift140* cKO mouse. G Examination of the efferent ductules of the *ift140* cKO mice. Upper panel: Normal efferent ductules histology was observed in the control mice. However, the lumen was highly dilated in *Ift140* cKO mice and more sperm accumulated here (arrows). Lower panel: Representative TEM images of sperm from efferent ductules of a *Ift140* cKO mouse. The arrows point to sperm axonemes with disrupted structure. H A cartoon to summarize the reproductive phenotypes observed in the absence of IFT140 motile cilia. Dysfunction of motile cilia in the efferent ductules in the *Ift140* cKO mice likely caused a failure of fluid absorption, which results in lumen dilation in both proximal efferent ductules and the seminiferous tubules. The accumulation of fluid inside the seminiferous tubules disrupted spermatogenesis. The formed sperm cells also accumulated at the proximal efferent ductules. Figure is adapted from images on opensource database (https://www.imaios.com/en/e-anatomy/anatomical-structures/body-of-epididymis-1541221040#) and SMART Servier medical art (https://smart.servier.com/)

## *Ift140* cKO mouse tracheal epithelial cells exhibit abnormally accumulated particles within cilia

Mutations in *Ift* genes are known to cause changes in cilia ultrastructure due to altered trafficking of ciliary proteins. Electron micrographs of control mTEC revealed typical ciliary structures (Fig. 7). In contrast, *Ift140* cKO mTEC exhibited the presence of electron-dense particles that appeared to accumulate within the cilium, as well as swollen tips in some cilia (Fig. 7, Supplemental Fig. 6) which is consistent with observations of IFT-B mutations in the motile cilia of *Chlamydomonas*, *Tetrahymena*, and the sensory cilia of *C. elegans* [46].

## Inactivation of *Ift140* in the motile cilia results in normal assembly of axonemal dynein motors, but impaired delivery of central pair apparatus proteins

Motile cilia normally have a “9 + 2” axonemal structure (Fig. 8A). Although *Ift140* cKO tracheal cilia are shorter than controls and have decreased cilia beat frequency, ultrastructural examination of the axoneme revealed morphologically normal appearing inner and outer dynein arms. However, the central pair apparatus appeared abnormal. Ultrastructural examination showed variation in central apparatus configurations, ranging from normal, to the absence of one or both microtubules (Fig. 8B). To investigate the assembly of components within the axonemes, localization of DNAI2 and DNAH2, components of outer and inner dynein arm motors, respectively, and SPAG16L (Chlamydomonas PF20) and SPAG17 (Chlamydomonas PF6), key components of the central pair apparatus were assessed by immunofluorescent microscopy. DNAI2 and DNAH2 were present in both control and *Ift140* cKO mice cilia even though these cilia were shorter (Fig. 8C, D). In contrast, SPAG16L and SPAG17 were present in control cilia, but in mutant mice most of the SPAG16L and SPAG17 proteins appeared to be localized in the cytoplasm of the cells (Fig. 8E, F).

## Discussion

IFT plays a significant role in ciliogenesis in *Chlamydomonas* and other ciliated species, as well as in primary cilia formation in vertebrates [4, 47, 48]. The elimination of IFT A component IFT140 in *Chlamydomonas* prevents cilia assembly [49]. The present study highlights that IFT140 is essential for motile ciliogenesis in mammals. *Ift140* cKO mice that survived to adulthood were completely infertile, and produce fewer, slow swimming sperm cells with abnormal sperm heads and shorter flagella together with ultrastructural defects. A similar phenotype was observed in the motile cilia in the respiratory tract. *Ift140* cKO mTEC exhibited abnormal cilia formation, characterized by reduced cilia number, a shorter length and decreased ciliary beat frequency compared to controls. Interestingly, in the brain ventricles, motile cilia appeared to be less affected, possible due to a different assembly program for these cells, indicating a need for further investigation of IFT in ependymal cilia.

IFT140 is a member of the IFT-A complex that has previously been studied primarily in non-mammalian organisms [50, 51]. We previously showed that IFT140 was required for sperm motility [28]. In our current studies, we extended these studies to show that IFT140 is also required for motile cilia function in other cell types. In airway cilia, we observed
<table><caption>**Table 1** Fertility and fecundity of the control and *Ift140<sup>flox/flox</sup>; FOXJ1-Cre* mutant mice</caption><thead><tr><th>Genotype</th><th>Male fertility</th><th>Litter size</th><th>Female fertility</th><th>Litter size</th></tr></thead><tbody><tr><td>Control</td><td>4/4</td><td>7.0±0.82 (n=4)</td><td>4/4</td><td>6.25±0.96 (n=4)</td></tr><tr><td>Ift140<sup>flox/flox</sup>; FOXJ1-Cre<sup>+/-</sup></td><td>0/3</td><td>0 (n=3) *</td><td>0/3</td><td>0 (n=3) *</td></tr></tbody></table>

Control includes: *Ift140<sup>flox/+</sup>; FOXJ1-Cre<sup>+/-</sup>*, *Ift140<sup>flox/+</sup>; FOXJ1-Cre<sup>-/-</sup>*, and *Ift140<sup>flox/flox</sup>; FOXJ1-Cre<sup>-/-</sup>*.

To test the fertility, four-month-old mice with the indicated genotypes were bred to four-month-old wild-type animals for two months. Litter sizes were recorded for each mating

abnormal particle accumulation and bulges along the ciliary shaft beneath the membrane, which are typical features of IFT-A mutations. We also found enlarged cilia tips,

attributed to failure of retrograde transport. The observed reduced cilia length in the mutant cilia has been typically attributed to IFT-B mutants owing to decreased delivery of

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<figure><img src="image_38.png" /><figcaption>**Fig. 2** *Ift140* cKO mice exhibited growth retardation and early death. A No difference was observed in one-day-old mice from all genotypes. A representative image of a one-day-old litter and the pups showing the same size in all genotypes. B A representative image of a two-week-old control mouse and *Ift140* cKO littermate. Notice that the size of the cKO mouse was much smaller than the control. C Body weight of one-week-old and two-week-old control and *Ift140*</figcaption></figure>

cKO mice. For both male (a) and female (b), the body weights were significantly lower in the cKO mice; the difference was greater at two-week-old. All cKO mice died before three weeks old after the second generation. D Morphology of the brains isolated from control and *Ift140* cKO mice. No obvious hydrocephalus was found in the *Ift140* cKO mice

<footnote>*P < 0.01</footnote>
<figure><img src="image_39.png" /><figcaption>**Fig. 3** Abnormal cilia formation in tracheal epithelial cells from *Ift140* cKO mice. A Immunofluorescence staining of trachea shows abnormal cilia in the *Ift140* cKO mice. The cilia signal is visualized by staining with anti-α-tubulin antibody. The cilia signal in the cKO mouse was dramatically reduced. B Histological examination of the trachea from control (upper) and *Ift140* cKO mice (lower).</figcaption></figure>

Fewer cilia were present in the cKO mice and the cilia appeared to be shorter (inserts). C Average length of the cilia is indicated in B. Three control and three cKO mice were analyzed. For each mouse, cilia were randomly selected from three regions, and the lengths were measured. \* P < 0.05

proteins during cilia assembly [52–55]. The mechanisms for inadequate cilia assembly leading to shorter cilia in the *Ift140* mutant is not known, but there are several potential interactions between components of IFT-A and IFT-B that may be disrupted [56].

The precise cargo handled by IFT140 is undefined. TEM of the short IFT140 mutant cilia suggests that the motor complexes for the inner and outer dynein arm do not require IFT140 (Fig. 8C, D). However, in the cilia cross sections the central apparatus ultrastructure appeared abnormal. Central apparatus protein SPAG16L and SPAG17 did not properly

localize in the cilia (Fig. 8E, F). It is also uncertain if there are specific IFT for trafficking central apparatus components such as SPAG16L. Immunofluorescence localization identified these proteins in the cytoplasm, which could be due to a transport or docking defect.

In the present investigation, only the first generation of homozygous mutant mice survived to adulthood. These mice did not exhibit any gross abnormalities, such as hydrocephalus. However, all these homozygous mutant mice were completely infertile. Subsequently homozygous mutant mice in all generations analyzed in this study died before reaching
<figure><img src="image_40.png" /><figcaption>**Fig. 4** Ciliogenesis in cultured tracheal epithelial cells from *Ift140* cKO mice is delayed. Tracheal epithelial cells were cultured and co-stained with anti-centrin antibodies to label basal bodies and anti-α-tubulin to label cilia. No difference was found in the number</figcaption></figure>

of centrin positive cells, however the α-tubulin signal was dramatically reduced in *Ift140* cKO mice. Left: representative images showing centrin and α-tubulin signals in the control and *Ift140* cKO mice; Right: Statistic analysis of the centrin and α-tubulin positive area

three weeks of age, accompanied by a significant reduction in body weight. A potential contributing factor could be the acquisition of additional epigenetic mutations [57]. Motile cilia are also present in the fetal esophagus at around week 16 of gestation but disappear by birth [58]. It is less likely that the *Ift140* mutant mice died an abnormal system because all the mutant mice were normal until one week after birth.

The observed spermatogenesis defects in *Ift-140 flox/flox*,*FOXJ1*-Cre± male mice are consistent with observations in a germ cell-specific *Ift140* knockout mice model [28]. This model revealed that IFT140 directly contributes to sperm flagella assembly, morphology and maintenance of normal sperm head morphology [28]. In addition to its direct impact on flagella assembly, another potential cause of aberrant spermatogenesis in the current *Ift140* cKO mice may stem from defects in the efferent duct. Mature spermatozoa produced in the seminiferous tubules pass through the efferent ducts, which reabsorb nearly 90% of the luminal fluids, during their transit to the epididymis. The efferent duct is lined with multiple motile ciliated cells, which are believed to play a crucial role in generating the luminal turbulence needed to prevent sperm aggregation [42–45, 59]. Disruption of motile cilia function in the efferent duct can lead to excessive fluid resorption, which leads to sperm aggregation and blockage of the ductal lumen. These ductal abnormalities cause fluid accumulation upstream and backflow into the seminiferous tubules, which results in seminiferous tubule dilation, epithelial degeneration and infertility. Although the inability to obtain adult *Ift140* cKO mice prevented further investigation, our findings demonstrate a significant reduction in cilia signals in the *Ift140* cKO mice.

It is hypothesized that the multiciliated cells in the efferent ducts of the *Ift140* cKO mice are defective, contributing to blockage of the efferent ducts, enlargement of the seminiferous tubule lumens, and dramatic reduction in cauda epididymal sperm concentrations. To investigate the cause of female infertility, attempts were made to collect reproductive organs from *Ift140* cKO mice. Unfortunately, due to technical issues, the attempts were not successful.

The perinatal defects and motile ciliopathies may also share some features with human ciliopathies whose features have strong primary cilia defects but may also involve motility defects. IFT-A genetic mutations are causative of human ciliopathy syndromes including short-rib thoracic dysplasia, cranioectodermal dysplasia, asphyxiating thoracic dystrophy, nephronophthisis, Senior-Loken syndromes, and retinitis pigmentosa. The high mortality of patients with these syndromes has made it difficult to study motile cilia in these syndromes, but many of them have clinical features of motile ciliopathies, especially respiratory symptoms [18, 60–62], perhaps dependent on the specific gene variant. Many ciliopathies affecting IFT and primary cilia also have a high perinatal mortality [58]. Because the *Ift140* cKO mice exhibited growth retardation and premature death, it will be difficult to determine if causes of death are similar to that in complex human ciliopathies.

While male and female fertility and respiratory cilia were affected, the motile ependymal cilia may be unaffected, based on the absence of hydrocephalus. Hydrocephalus, characterized by the aberrant flow of cerebrospinal fluid, is another phenotype associated with motile cilia defects that is common in mice deficient in motile cilia genes but very rare in humans with PCD [40, 63]. The *Ift140* cKO mice

<figure><img src="image_41.png" /></figure>
<figure><img src="image_42.png" /><figcaption>**Fig. 5** Reduced cilia length in tracheal epithelial cells from the *ift140* cKO mice. A Examination of cilia length by immunofluorescence staining. Left: representative images of the staining results; right: Statistical analysis of cilia length measurements from the images. B Exam-</figcaption></figure>

ination of cilia length by TEM. Left: representative TEM images; right: Statistic analysis of cilia length as evaluated from the TEM images

did not exhibit any signs of gross hydrocephalus. Immunofluorescence staining also indicated that the cilia length in the brain ventricle epithelial cells appeared to be unaffected in the *IFT140* cKO (Supplemental Fig. 2). It is possible that IFT140 does not play a major role in motile ciliogenesis in brain ventricle epithelial cells.

The *IFT140* cKO also lacked evidence of developmental situs abnormalities. Left-right axis patterning has been established to be associated with *Foxj1* expression and the regulation of asymmetric genes in the embryonic nodal cells [30, 34, 64]. A recent study demonstrated that IFT140 was required for left-right patterning during early embryogenesis using a global Cre-driver [64]. One possibility is that the few affected mice in our model died at embryonic stage; another possibility is that our Cre line

does not efficiently delete *ift140* in the nodal cells. It is also possible that the number of animals analyzed in this study was relatively low and there were no mice with situs inversus by chance. The absence of left-right defects in the cKO mice may more likely be related to the role of IFT140 for central apparatus function, as suggested by loss of SPAG16L decreased central apparatus structures observed in sperm of our *ift140* cKO mouse. The "9 + 0" motile cilia in nodal cells generate a leftward fluid flow through spinning, which serves as the primary signal for establishing left-right axis asymmetry [30, 34, 64–67]. In PCD and mouse models, gene mutations affecting the central pair do not result in left-right asymmetry defects [68].

Based on the observations in the present study, IFT140 is essential for motile ciliogenesis in selected organs with
<figure><img src="image_43.png" /><figcaption>**Fig. 7** Abnormal protein trafficking in the cultured tracheal epithelial cells (mTEC) *from Ift140* cKO. TEM was conducted in cultured mTEC from the control and *Ift140* cKO mice. The mTEC cells from the control mice showed long and smooth cilia (left). However, in the</figcaption></figure>

cells from the *Ift140* cKO mice, the cilia were not only shorter, they trapped particles and bulges (arrowheads). In some cilia, the tips were swollen (arrows), a typical feature of defective IFT

motile cilia. It is possible that short cilia with diminished cilia beat frequency are a feature of acquired ciliopathies, particularly attributed to cigarette smoke exposure, perhaps due to interruption of IFT proteins including IFT140 [69]. This study demonstrates that IFT not only plays a role in primary cilia formation but is also crucial for motile ciliogenesis in mammals. It also contributes in to identification the common and specific IFT molecular actors within the diverse cilia types. Even though we identified SPAG16L and SPAG17 to be potential cargos of IFT140 in assembling the motile cilia, due to technique limitations, we were not able to discover more targets in this study. With more sensitive proteomics approaches available, it becomes possible to dissect the IFT140 complex

globally in these motile cilia. For example, Wallingford's lab studied the IFT122 complex using the powerful DIF-FRAC method [70]. The same method could be used to investigate the role of IFT140 and other IFT complexes in cilia assembly. Using affinity purification coupled with mass spectrometry, IFT140 interaction in HEK293 cells was also identified [71]. Normal IFT-A complex structure was also dissected by cryo-EM [51] as well as combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold 2-based prediction [72]. Future studies of mutant IFT cilia using these methods will be instructive. In conclusion, our studies enriched the current knowledge in the IFT-A assembly and IFT train
<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

**Fig. 8** Inactivation of *Ift140* in motile cilia does not change the axonemal “9 + 2” structure, but affects the localization of the key central pair apparatus. **A** Cartoon showing normal representative motile cilia structure; **B** Cilia in *Ift140* cKO mTEC retain normal “9 + 2” axonemal structure as examined by TEM; **C** Outer dynein arm protein DNAI2 localization in TEC of control and *Ift140* cKO mice. In control mice, DNAI2 signal was present in the cilia. In the *Ift140* cKO mice, even though less cilia were present, DNAI2 signal remained in the cilia; **D** Inner dynein arm protein DNAH2 localization in TEC of control and *Ift140* cKO mice. In control mice,

DNAH2 signal was present in the cilia. In the *Ift140* cKO mice, even though less cilia were present, DNAH2 also in the cilia; **E** Central pair protein SPAG16L localization in mTEC of control and *Ift140* cKO mice. In control mice, SPAG16L signal was present in the cilia. In the *Ift140* cKO mice, SPAG16L signal was largely present in the cytoplasm; **F** Central pair protein SPAG17 localization in mTEC of control and *Ift140* cKO mice. In control mice, SPAG17 signal was present in the cilia. In the *Ift140* cKO mice, SPAG17 signal was largely present in the cytoplasm

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>
formation and can serve as a basis for future structural-
function analyses.

**Materials and methods**

Generation of Ift140 flox/flox; FOXJ1-Cre mouse model

Floxed Ift140 mice were generated by Dr. Gregory J. Pazour, University of Massachusetts Medical School [73]. Transgenic mouse lines, expressing improved Cre recombinase under the control of a 1.2 kilobase promoter region of the human Forkhead box J1 (FOXJ1) gene, were obtained from Dr. Michael J. Holtzman, Washington University School of Medicine in St. Louis [41]. Three to four-month-old *FOXJ1-Cre* males were crossed with three to four-month-old *Ift140* females to obtain *Ift140<sup>flox/+</sup>*; *FOXJ1-Cre<sup>±</sup>* mice. Three to four-month-old *Ift140<sup>flox/+</sup>*; *FOXJ1-Cre<sup>±</sup>* males were backcrossed with three- to four-month-old *Ift140<sup>flox/flox</sup>* females again, and the *Ift140<sup>flox/flox</sup>*; *FOXJ1-Cre<sup>±</sup>* were considered to be the homozygous knockout mice (cKO), and *Ift140<sup>flox/+</sup>*; *FOXJ1-Cre<sup>±</sup>* mice were used as the controls. Mice were genotyped by PCR using multiplex PCR mix (Bioline, Cat No. BIO25043) and primers as indicated in the Supplemental Table 1. The presence of the *FOXJ1-Cre* allele was evaluated as described in Zhang et al. [41], and *Ift140* genotypes were determined as described previously [64].

Tissue histology

Testis, epididymides, trachea, and other indicated tissues
were fixed in 4% paraformaldehyde overnight. The tissues
were embedded in paraffin, sectioned at 5 µm thickness,
deparaffined, and stained with hematoxylin and eosin,
using standard procedures. Slides were examined using
a BX51 Olympus microscope (Olympus Corp., Melville,
NY, Center Valley, PA), and photographs were taken with
a ProgRes C14 camera (Jenoptik Laser, Germany).

Mouse tracheal epithelial cell culture

Progenitor basal cells were isolated from the trachea from
3-4-week-old wild type, heterozygous, and *Ift140* cKO
mice following incubation with pronase for 24 h, at 4 °C.
Progenitor basal cells were expanded on collagen-coated
supported membranes (Transwell, Corning, Cat. no. 3460)
in custom made growth factor-containing media, with the
additional use of dual SMAD inhibition over 5-7 days as
described [37, 74]. Once confluent, the apical media was

removed and cells were differentiated at air-liquid inter-
face on Transwell membranes for up to four weeks prior
to assay [75].

Cilia beat frequency measurement

CBF was quantified as described by Horani et al., using the
Sisson–Ammons Video Analysis (SAVA, Ammons Engi-
neering) [37]. All cells that had visible and in-focus full
length of cilia were considered for cilia length measure-
ments. CBF was recorded in: (1) tracheas isolated from
mice and the CBF was analyzed immediately; (2) tracheae
isolated and shipped overnight in DMEM with 10% fetal
calf serum to the Brody laboratory, and after transfer to
mTEC media and placement tissue culture incubator; (3)
cultured mTEC.

To quantify CBF from each type of cell preparations,
cilia were imaged using a Nikon Ti inverted microscope
enclosed in an environmental chamber held at 37 °C. Data
were recorded and averaged using Sisson–Ammons Video
Analysis software (SAVA, Ammons Engineering, Clio, MI)
[37]. To record CBF of cells from tracheae, rings cut from
trachea and cells scraped from the tracheal wall were placed
on a glass microscope slide with mouse trachea culture
media under a coverslip. Beat frequency was recorded from
cell clusters containing ciliated cells using a 40X Hoffman
modulation contrast lens (NAMC3, Nikon) or 20X phase
contrast lens. CBF from each cluster was averaged and the
data from each cluster was considered as a single data sam-
ple. CBF of mTEC cultures was quantified from recordings
of five fields per Transwell insert using at 20X phase objec-
tive. The average CBF of each field was recorded as a single
data point.

Immunofluorescence staining

The indicated tissues from the control and cKO mice were
fixed with 4% paraformaldehyde in 0.1 M phosphate buff-
ered saline (PBS; pH 7.4), and 5 µm paraffin sections were
made. Cultured mTEC were immunostained as previously
described. The sections or cells were incubated with the
indicated primary antibodies at 4 °C overnight. SPAG16L
(1:300 dilution) and SPAG17 (1:300 dilution) antibod-
ies were previously generated in our laboratory [76, 77].
Mouse acetylated alpha tubulin antibodies were purchased
from Sigma-Aldrich (clone 6-11B-1, T6793. 1:300 dilution)
and Proteintech (66,200–1-ig. 1:300 dilution). Slides were
washed with PBS and incubated for 1 h at room tempera-
ture with Alexa 488-conjugated anti-mouse IgG secondary
antibody (1:1000; Jackson ImmunoResearch Laboratories)
or Cy3-conjugated anti-rabbit IgG secondary antibody
(1:1000; Jackson ImmunoResearch Laboratories). Following
secondary antibody incubation, the slides were washed three
times with PBS, and mounted using VectaMount with DAPI
(Vector Laboratories, Burlingame, USA), and sealed with a
cover slip. Images were captured by confocal laser-scanning
microscopy (Zeiss LSM 700). Some fluorescence images
were captured using a spinning disc confocal system (Andor
Dragonfly 200) mounted to an inverted microscope (Leica
DMI8) with a 63X oil-immersion objective (Leica HC PL
APO, NA 1.4) and a sCMOS camera (Andor Zyla). Consistent
acquisition settings were applied across all color channels,
and brightness/contrast adjustments were uniformly
applied in ImageJ for display.

For detection of inner and outer dynein arm motor pro-
teins in mouse lung, formalin-fixed, paraffin embedded tis-
sues were sectioned and subjected to antigen retrieval by
submersion in antigen retrieval buffer (Antigen unmask-
ing solution, Vector, H-3300) and heating under pressure
(Decloaking chamber, BioCare Medical) for 5 min, then
cooled to room temperature. Tissue sections were incu-
bated in blocking buffer (bovine serum albumin, 3%, in
PBS) for 30 min at room temperature, then incubated in
blocking buffer with the following primary antibodies at
4 °C overnight: Acetylated alpha-tubulin (1:5000, mouse
clone 6-11B-1), DNAI2 (1:200, rabbit, Proteintech, Cat. no.
17533), DNAH2 (1:2000; Sigma, HPA067103). Slides were
washed and tissues were incubated with secondary antibod-
ies (Donkey anti-rabbit or Donkey anti-mouse with Alexa
Fluor 488 or 555, Life Technologies) for 30 min at room
temperature. Tissue sections were mounted in medium con-
taining DAPI (Flouroshield, Sigma). Images were captured
using a Leica 6000 microscope equipped with a cooled digi-
tal camera and Leica imaging software (LAS X).

Electron microscopy

mTEC used for transmission electron microscopy analysis
were cultured in Transwell filters and prepared as previously
described by Horani et al. [37]. Briefly, cells were fixed with
mix of 2% paraformaldehyde plus 2% glutaraldehyde in 100
mM sodium cacodylate buffer overnight at 4 °C. Cells were
post fixed with osmium tetroxide (1%, Ted Pella, 18,459),
stained in uranyl acetate (1%, Electron Microscopy Sci-
ences, 22,400–1), embedded in Eponate 12 resin (Ted Pella,
18,005). Sections (95 nm) were imaged on grids using a
JOEL 1200 EX TEM.

Cilia length measurement

Cilia length was measured in fixed mTEC cultures that were
immunostained and parallel samples prepared for transmis-
sion electron microscopy. In all samples, cilia length was
measured by image J (FIJI) using the distant measurement
tool. Cells cultured on Transwell membranes were fixed

and immunostained for acetylated alpha-tubulin, scraped
from membranes and mounted under coverslips. All cells
that had visible and the full length of cilia in focus were
considered for cilia length measurements. A total of 40–50
cilia in multiple fields were measured for control (WT
and *Ift140<sup>+/flox</sup>; *FOXI1-Cre<sup>±</sup>*) and *Ift140<sup>deficient</sup> (*Ift-
140<sup>flox/flox</sup>; *FOXI1-Cre<sup>±</sup>*) genotypes. Cilia length was meas-
ured from 17 to 19 different electron micrographs of the
same genotype.

**Statistical analysis**

Graphs were created using Microsoft Excel and GraphPad Prism. Statistical analyses were performed using Student's *t* test. P > 0.05 was considered as not significant and by convention *p* < 0.05, **P** < 0.01. Multiple groups were compared using the Kruskal–Wallis test and Dunn's multiple comparison test.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00018-025-05710-z.

**Acknowledgements** This research was supported by Wayne State University Start-up fund, NIH grants HD105944 (ZZ), HD107579 (ZZ), and HD114311 (ZZ). Steven L. Brody is supported by NIH HL146601, HL128370, and the Barnes-Jewish Hospital Foundation. Aminata Touré is funded by INSERM, CNRS, Université Grenoble Alpes and the Agence Nationale pour la Recherche (ANR-17-CE13-0023 DIVERCIL, ANR-19-CE17-0014 FLAGELOME). Gregory J. Pazour is supported by NIH grant GM060992. Michael Holtzman is supported by NIH R35 HL145242.

**Author contributions** Y. Yap, J. Pan, J. Xu, S. Yuan, C. Niu, C. Zheng, W. Li, T. Zhou, T. Li, and Y. Zhang performed research; M. Holtzman, G. Pazour, R. Hess, C. Kelly, and A. Touré contributed reagents; S. Brody and Z Zhang wrote the paper.

**Funding** Foundation for the National Institutes of Health, HD105944, Zhibing Zhang, HD114311, Zhibing Zhang, HL146601, Steven L. Brody, HL128370, Steven L. Brody, GM060992, Gregory J. Pazour, HL-145242, Michael J. Holtzman, Agence Nationale de la Recherche, ANR-17-CE13-0023 DIVERCIL, Aminata Touré, ANR-19-CE17-0014 FLAGELOME, Aminata Touré.

**Data availability** The data is available from the corresponding author upon reasonable request.

Declarations

**Conflict of interest** There is no conflict of interest that could be per-
ceived as prejudicing the impartiality of the research reported. The
authors declare no competing interests.

**Ethics statement** All animal research was executed in compliance with the guidelines of the Wayne State University Institutional Animal Care with the Program Advisory Committee (Protocol number: 18-02-0534).

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
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References

1. Satir P, Christensen ST (2008) Structure and function of mammalian cilia. Histochem Cell Biol 129:687-693

2. Mohri H, Inaba K, Ishijima S, Baba SA (2012) Tubulin-dynein system in flagellar and ciliary movement. Proc Jpn Acad Ser B Phys Biol Sci 88(8):397-415

3. Hao K, Chen Y, Yan X, Zhu X (2021) Cilia locally synthesize proteins to sustain their ultrastructure and functions. Nat Commun 12(1):6971

4. Kozminski KG, Johnson KA, Forscher P, Rosenbaum JL (1993) A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad Sci USA 90:5519-5552

5. Hao L, Acar S, Evans J, Ou G. Scholey JM (2009) Analysis of intraflagellar transport in *C. elegans* sensory cilia. Methods Cell Biol 93:235-266

6. Man YH, Warmbrunn I, Zhang L, Zhang ZB (2022) The roles of intraflagellar transport (IFT) protein 25 in mammalian signaling transduction and flagellogenesis. Asian J Androl 24(3):238-242

7. Taschner M, Lorentzen E (2016) The intraflagellar transport machinery. Cold Spring Harb Perspect Biol 8(10):a028092

8. Rosenbaum JL, Witman GB (2002) Intraflagellar transport. Nat Rev Mol Cell Biol 3(11):813-825

9. Scholey JM (2008) Intraflagellar transport motors in cilia: moving along the cell's antenna. J Cell Biol 180(1):23-29

10. Scholey JM (2013) Kinesin-2: a family of heterotrimeric and homodimeric motors with diverse intracellular transport functions. Annu Rev Cell Dev Biol 29:443-469

11. Hou Y, Witman GB (2015) Dynein and intraflagellar transport. Exp Cell Res 334(1):26-34

12. Aldahmesh MA, Li Y, Alhashem A, Anazi S, Alkuraya H, Hashem M, Awaji AA, Sogaty S, Alkharashi A, Alzahrani S, Al Hazzaa SA, Xiong Y, Kong S, Sun Z, Alkuraya FS (2014) IFT27, encoding a small GTPase component of IFT particles, is mutated in a consanguineous family with Bardet-Biedl syndrome. Hum Mol Genet 23(12):3307-3315

13. Arts HH, Bongers EM, Mans DA, van Beersum SE, Oud MM, Bolat E, Spruijt L, Cornelissen EA, Schuurs-Hoeijmakers JH, de Leeuw N, Cormier-Daire V, Brunner HG, Knoers NV, Roepman R (2011) C14ORF179 encoding IFT43 is mutated in Sensenbrenner syndrome. J Med Genet 48(6):390-395

14. Gilissen C, Arts HH, Hoischen A, Spruijt L, Mans DA, Arts P, van Lier B, Steehouwer M, van Reeuwijk J, Kant SG, Roepman R, Knoers NV, Veltman JA, Brunner HG (2010) Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome. Am J Hum Genet 87(3):418-423

15. Perrault I, Saunier S, Hanein S, Filhol E, Bizet AA, Collins F, Salih MA, Gerber S, Delphin N, Bigot K, Orssaud C, Silva E, Baudouin V, Oud MM, Shannon N, Le Merrer M, Roche O, Pietrement C, Goumid J, Baumann C,Bole-Feysot C, Nitschke P, Zahrate M, Beales P, Arts HH, Munnich A, Kaplan J, Antignac C, Cormier-Daire V, Rozet JM (2012) Mainzer-saldino syndrome

is a ciliopathy caused by IFT140 mutations. Am J Hum Genet
90(5):864-870

16. Bredrup C, Saunier S, Oud MM, Fiskerstrand T, Hoischen A, Brackman D, Leh SM, Midtbø M, Filhol E, Bole-Feysot C, Nitschké P, Gilissen C, Haugen OH, Sanders JS, Stolte-Dijkstra I, Mans DA, Steenbergen EJ, Hamel BC, Matignon M, Pfundt R, Jeanpierre C, Boman H, Rødahl E, Veltman JA, Knappskog PM, Knoers NV, Roepman R, Arts HH (2011) Ciliopathies with skeletal anomalies and renal insufficiency due to mutations in the IFT-A gene WDR19. Am J Hum Genet 89(5):634-643

17. Schmidts M, Frank V, Eisenberger T, Al Turki S, Bizet AA, Antony D, Rix S, Decker C, Bachmann N, Bald M, Vinke T, Toenshoff B, Di Donato N, Neuhann T, Hartley JL, Maher ER, Bogdanovic R, Peco-Antic A, Mache C, Hurles ME, Joksic I, Guc Scekic M, Dobricic J, Brankovic-Magic M, Bolz HJ, Pazour GJ, Beales PL, Scambler PJ, Saunier S, Mitchison HM, Bergmann C (2013) Combined NGS approaches identify mutations in the intra flagellar transport gene IFT140 in skeletal ciliopathies with early progressive kidney disease. Hum Mutat 34:714-724

18. Xu M, Yang L, Wang F, Li H, Wang X, Wang W, Ge Z, Wang K, Zhao L, Li H, Li Y, Sui R, Chen R (2015) Mutations in human IFT140 cause non-syndromic retinal degeneration. Hum Genet 134:1069-1078

19. Wang X, Sha YW, Wang WT, Cui YQ, Chen J, Yan W, Hou XT, Mei LB, Yu CC, Wang J (2019) Novel IFT140 variants cause sper matogenic dysfunction in humans. Mol Genet Genomic Med 7:e920

20. Despotes KA, Zariwala MA, Davis SD, Ferkol TW (2024) Pri-
mary ciliary dyskinesia: a clinical review. Cells 13(11):97

21. Senum SR, Li YSM, Benson KA, Joli G, Olinger E, Lavu S, Mad-
sen CD, Gregory AV, Neatu R, Kline TL, Audrezet MP, Outeda
P, Nau CB, Meijer E, Ali H, Steinman TI, Mrug M, Phelan PJ,
Watnick TJ, Peters DJM, Ong ACM, Conlon PJ, Perrone RD,
Cornec-Le Gall E, Hogan MC, Torres VE, Sayer JA, Genomics
England Research Consortium, the HALT PKD, CRISP, DIPAK,
ADPKD Modifier, and TAME PKD studies, Harris PC (2022)
Monoallelic IFT140 pathogenic variants are an important cause
of the autosomal dominant polycystic kidney-spectrum phenotype.
Am J Hum Genet 109:136-156

22. Bakey Z, Cabrera OA, Hoefele J, Antony D, Wu K, Stuck MW, Micha D, Eguether T, Smith AO, van der Wel NN, Wagner M, Strittmatter L, Beales PL, Jonassen JA, Thiffault I, Cadieux-Dion M, Boyes L, Sharif S, Tüysüz B, Dunstheimer D, Niessen HWM, Devine W, Lo CW, Mitchison HM, Schmidts M, Pazour GJ (2023) IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans. PLoS Genet 19(6):e1010796

23. Gilley SK, Stenbit AE, Pasek RC, Sas KM, Steele SL, Amria M, Bunni MA, Estell KP, Schwiebert LM, Flume P, Gooz M, Haycraft CJ, Yoder BK, Miller C, Pavlik JA, Turner GA, Sisson JH, Bell PD (2014) Deletion of airway cilia results in noninflam-
matory bronchiectasis and hyperreactive airways. Am J Physiol
Lung Cell Mol Physiol 306(2):L162-L169

24. Zhang Z, Li W, Zhang Y, Zhang L, Teves ME, Liu H, Strauss JF 3rd, Pazour GJ, Foster JA, Hess RA, Zhang Z (2016) Intraflagellar transport protein IFT20 is essential for male fertility and spermiogenesis in mice. Mol Biol Cell 27(23):3705-3716

25. Liu H, Li W, Zhang Y, Zhang Z, Shang X, Zhang L, Zhang S, Li Y, Somoza AV, Delpi B, Gerton GL, Foster JA, Hess RA, Pazour GJ, Zhang Z (2017) IFT25, an intraflagellar transporter protein dispensable for ciliogenesis in somatic cells, is essential for sperm flagella formation. Biol Reprod 96(5):993-1006

26. Zhang Y, Liu H, Li W, Zhang Z, Shang X, Zhang D, Li Y, Zhang S, Liu J, Hess RA, Pazour GJ, Zhang Z (2017) Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is

<figure><img src="image_50.png" /></figure>
essential for male fertility and spermiogenesis in mice. Dev Biol
432(1):125-139

27. Qu W, Yuan S, Quan C, Huang Q, Zhou Q, Yap Y, Shi L, Zhang D, Guest T, Li W, Yee SP, Zhang L, Cazin C, Hess RA, Ray PF, Kherraf ZE, Zhang Z (2020) The essential role of intraflagellar transport protein IFT81 in male mice spermiogenesis and fertility. Am J Physiol Cell Physiol 318(6):C1092-C1106

28. Zhang Y, Liu H, Li W, Zhang Z, Zhang S, Teves ME, Stevens C, Foster JA, Campbell GE, Windle JJ, Hess RA, Pazour GJ, Zhang Z (2018) Intraflagellar transporter protein 140 (IFT140), a component of IFT-A complex, is essential for male fertility and spermiogenesis in mice. Cytoskeleton 75(2):70-84

29. Blatt EN, Yan XH, Wuerffel MK, Hamilos DL, Brody SL (1999) Forkhead transcription factor HFH-4 expression is temporally related to ciliogenesis. Am J Respir Cell Mol Biol 21(2):168-176

30. Brody SL, Yan XH, Wuerffel MK, Song SK, Shapiro SD (2000) Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. Am J Respir Cell Mol Biol 23(1):45-51

31. Hackett BP, Brody SL, Liang M, Zeitz ID, Bruns LA, Gitlin JD (1995) Primary structure of hepatocyte nuclear factor/forkhead homologue 4 and characterization of gene expression in the developing respiratory and reproductive epithelium. Proc Natl Acad Sci USA 92(10):4249-4253

32. Lim L, Zhou H, Costa RH (1997) The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo. Proc Natl Acad Sci USA 94(7):3094-3099

33. Yu X, Ng CP, Habacher H, Roy S (2008) Foxj1 transcription factors are master regulators of the motile ciliogenic program. Nat Genet 40(12):1445-1453

34. Chen J, Knowles HJ, Hebert JL, Hackett BP (1998) Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry. J Clin Invest 102(6):1077-1082

35. Olbrich H, Häffner K, Kispert A, Völkel A, Volz A, Sasmaz G, Reinhardt R, Hennig S, Lehrach H, Konietzko N, Zariwala M, Noone PG, Knowles M, Mitchison HM, Meeks M, Chung EM, Hildebrandt F, Sudbrak R, Omran H (2002) Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left-right asymmetry. Nat Genet 30(2):143-144

36. Ostrowski LE, Yin W, Rogers TD, Busalacchi KB, Chua M, O'Neal WK, Grubb BR (2010) Conditional deletion of dnaic1 in a murine model of primary ciliary dyskinesia causes chronic rhinosinusitis. Am J Respir Cell Mol Biol 43(1):55-63

37. Horani A, Gupta DK, Xu J, Xu H, Carmen Puga-Molina LD, Santi CM, Ramagiri S, Brennan SK, Pan J, Koenitzer JR, Huang T, Hyland RM, Gunsten SP, Tzeng SC, Strahle JM, Mill P, Mahjoub MR, Dutcher SK, Brody SL (2023) The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes. JCI Insight 8(11):e168836

38. Becker-Heck A, Zohn IE, Okabe N, Pollock A, Lenhart KB, Sullivan-Brown J, McSheene J, Loges NT, Olbrich H, Haeffner K, Fliegauf M, Horvath J, Reinhardt R, Nielsen KG, Marthin JK, Baktai G, Anderson KV, Geisler R, Niswander L, Omran H, Burdine RD (2011) The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation. Nat Genet 43(1):79-84

39. Abdelhamed Z, Vuong SM, Hill L, Shula C, Timms A, Beier D, Campbell K, Mangano FT, Stottmann RW, Goto J (2018) A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice. Development 145(1):dev154500

40. Lee L, Ostrowski LE (2021) Motile cilia genetics and cell biology: big results from little mice. Cell Mol Life Sci 78(3):769-797

41. Zhang Y, Huang G, Shornick LP, Roswit WT, Shipley JM, Brody SL, Holtzman MJ (2007) A transgenic FOXJ1-Cre system for

gene inactivation in ciliated epithelial cells. Am J Respir Cell Mol
Biol 36(5):515-519

42. Hess RA (2025) Efferent ductules: structure and function, reference module in biomedical sciences: encyclopedia of reproduction, 3rd edn. Elsevier

43. Danielian PS, Hess RA, Lees JA (2016) E2f4 and E2f5 are essential for the development of the male reproductive system. Cell Cycle 15(2):250-260

44. Hoque M, Chen D, Hess RA, Li FQ, Takemaru KI (2021) CEP164 is essential for efferent duct multiciliogenesis and male fertility. Reproduction 162(2):129-139

45. Yuan S, Liu Y, Peng H, Tang C, Hennig GW, Wang Z, Wang L, Yu T, Klukovich R, Zhang Y, Zheng H, Xu C, Wu J, Hess RA, Yan W (2019) Motile cilia of the male reproductive system require miR-34/miR-449 for development and function to generate luminal turbulence. Proc Natl Acad Sci USA 116(9):3584-3593

46. Ishikawa H, Marshall WF (2017) Intraflagellar transport and ciliary dynamics. Cold Spring Harb Perspect Biol 9(3):a021998

47. Silflow CD, Lefebvre PA (2001) Assembly and motility of eukaryotic cilia and flagella: lessons from *Chlamydomonas reinhardtii*. Plant Physiol 127(4):1500-1507

48. Orozco JT, Wedaman KP, Signor D, Brown H, Rose L, Scholey JM (1999) Movement of motor and cargo along cilia. Nature 398(6729):674

49. Picariello T, Brown JM, Hou Y, Swank G, Cochran DA, King OD, Lechtreck K, Pazour GJ, Witman GB (2019) A global analysis of IFT-A function reveals specialization for transport of membrane-associated proteins into cilia. J Cell Sci 132(3):jcs220749

50. Engel BD, Ishikawa H, Wemmer KA, Geimer S, Wakabayashi K, Hirono M, Craige B, Pazour GJ, Witman GB, Kamiya R, Marshall WF (2012) The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function. J Cell Biol 199(1):151-167

51. Jiang M, Palicharla VR, Miller D, Hwang SH, Zhu H, Hixson P, Mukhopadhyay S, Sun J (2023) Human IFT-A complex structures provide molecular insights into ciliary transport. Cell Res 33(4):288-298

52. Iomini C, Babaev-Khaimov V, Sassaroli M, Piperno G (2001) Protein particles in *Chlamydomonas* flagella undergo a transport cycle consisting of four phases. J Cell Biol 153:13-24

53. Iomini C, Li L, Esparza JM, Dutcher SK (2009) Retrograde intraflagellar transport mutants identify complex A proteins with multiple genetic interactions in *Chlamydomonas reinhardtii*. Genetics 183:885-896

54. Efimenko E, Blacque OE, Ou G, Haycraft CJ, Yoder BK, Scholey JM, Leroux MR, Swoboda P (2006) *Caenorhabditis elegans* DYF-2, an orthologue of human WDR19, is a component of the intraflagellar transport machinery in sensory cilia. Mol Biol Cell 17:4801-4811

55. Tsao C-C, Gorovsky MA (2008) Tetrahymena IFT122A is not essential for cilia assembly but plays a role in returning IFT proteins from the ciliary tip to the cell body. J Cell Sci 121:428-436

56. Webb S, Mukhopadhyay AG, Roberts AJ (2020) Intraflagellar transport trains and motors: insights from structure. Semin Cell Dev Biol 107:82-90

57. Peaston AE, Whitelaw E (2006) Epigenetics and phenotypic variation in mammals. Mamm Genome 17(5):365-374

58. Ménard D, Arsenault P (1987) Maturation of human fetal esophagus maintained in organ culture. Anat Rec 217(4):348-354

59. Hess RA (2015) Small tubules, surprising discoveries: From efferent ductules in the turkey to the discovery that estrogen receptor alpha is essential for fertility in the male. Anim Reprod 12(1):7-23

60. Moalem S, Keating S, Shannon P, Thompson M, Millar K, Nykamp K, Forster A, Noor A, Chitayat D (2013) Broadening the ciliopathy spectrum: motile cilia dyskinesia, and nephronophthisis
associated with a previously unreported homozygous mutation in the INVS/NPHP2 gene. Am J Med Genet A 161(7):1792-1796

61. Boerwinkle C, Marshall JD, Bryant J, Gahl WA, Olivier KN, Gunay-Aygun M (2017) Respiratory manifestations in 38 patients with Alström syndrome. Pediatr Pulmonol 52(4):487-493

62. Duran I, Taylor SP, Zhang W, Martin J, Qureshi F, Jacques SM, Wallerstein R, Lachman RS, Nickerson DA, Bamshad M, Cohn DH, Krakow D (2017) Mutations in IFT-A satellite core component genes *IFT43* and *IFT121* produce short rib polydactyly syndrome with distinctive campomelia. Cilia 6:7

63. Hagenlocher C, Walentek P, Müller C, Thumberger T, Feistel K (2013) Ciliogenesis and cerebrospinal fluid flow in the developing Xenopus brain are regulated by foxj1. Cilia 2(1):12

64. Zhang M, Bolfing MF, Knowles HJ, Karnes H, Hackett BP (2004) Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice. Biochem Biophys Res Commun 324(4):1413-1420

65. Francis RJB, San Agustin JT, Szabo Rogers HL, Cui C, Jonassen JA, Eguether T, Follit JA, Lo CW, Pazour GJ (2023) Autonomous and non-cell autonomous role of cilia in structural birth defects in mice. PLoS Biol 21(12):e3002425

66. Sulik K et al (1994) Morphogenesis of the murine node and notochordal plate. Dev Dyn 201(3):260-278

67. Hirokawa N, Tanaka Y, Okada Y, Takeda S (2006) Nodal flow and the generation of left-right asymmetry. Cell 125(1):33-45

68. Wallmeier J, Nielsen KG, Kuehni CE, Lucas JS, Leigh MW, Zariwala MA, Omran H (2020) Motile ciliopathies. Nat Rev Dis Primers 6(1):77

69. Hessel J, Heldrich J, Fuller J, Staudt MR, Radisch S, Hollmann C, Harvey BG, Kaner RJ, Salit J, Yee-Levin J, Sridhar S, Pillai S, Hilton H, Wolff G, Bitter H, Visvanathan S, Fine J, Stevenson CS, Crystal RG, Tilley AE (2014) Intraflagellar transport gene expression associated with short cilia in smoking and COPD. PLoS ONE 9(1):e85453

70. Leggere JC, Hibbard JVK, Papoulas O, Lee C, Pearson CG, Marcotte EM, Wallingford JB (2024) Label-free proteomic

comparison reveals ciliary and nonciliary phenotypes of IFT-A mutants. Mol Biol Cell 35(3):ar39

71. Beyer T, Diwan GD, Leonhard T, Dahlke K, Klose F, Stehle IF, Seda M, Bolz S, Woerz F, Russell RB, Jenkins D, Ueffing M, Boldt K (2025) Ciliopathy-associated missense mutations in IFT140 are tolerated by the inherent resilience of the IFT machinery. Mol Cell Proteom 24(3):100916

72. McCafferty CL et al (2022) Integrative modeling reveals the molecular architecture of the intraflagellar transport A (IFT-A) complex. Elife 11:e81977

73. Jonassen JA, SanAgustin J, Baker SP, Pazour GJ (2012) Disruption of IFT complex A causes cystic kidneys without mitotic spindle misorientation. J Am Soc Nephrol 23(4):641-651

74. Mou H, Vinarsky V, Tata PR, Brazauskas K, Choi SH, Crooke AK, Zhang B, Solomon GM, Turner B, Bihler H et al (2016) Dual SMAD signaling inhibition enables long-term expansion of diverse epithelial basal cells. Cell Stem Cell 19:217-231

75. You Y, Richer EJ, Huang T, Brody SL (2002) Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population. Am J Physiol Lung Cell Mol Physiol 283:1315-1321

76. Zhang Z, Sapiro R, Kapfhamer D, Bucan M, Bray J, Chennathukuzhi V, McNamara P, Curtis A, Zhang M, Blanchette-Mackie EJ, Strauss JF 3rd (2002) A sperm-associated WD repeat protein orthologous to Chlamydomonas PF20 associates with Spag6, the mammalian orthologue of Chlamydomonas PF16. Mol Cell Biol 22(22):7993-8004

77. Zhang Z, Jones BH, Tang W, Moss SB, Wei Z, Ho C, Pollack M, Horowitz E, Bennett J, Baker ME, Strauss JF 3rd (2005) Disconnecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16. Mol Cell Proteom 4(7):914-923

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## Authors and Affiliations

Yi Tian Yap¹ · Jiehong Pan² · Jian Xu² · Shuo Yuan¹,³ · Changmin Niu¹,⁴ · Cheng Zheng¹,³ · Wei Li¹ · Ting Zhou¹,³ · Tao Li¹,⁵ · Yong Zhang² · Michael J. Holtzman² · Gregory J. Pazour⁶ · Rex A. Hess⁷ · Christopher V. Kelly⁸ · Aminata Touré⁹ · Steven L. Brody² · Zhibing Zhang¹,¹⁰

<figure><img src="image_51.png" /></figure>

Zhibing Zhang
gn6075@wayne.edu

1 Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI 48201, USA

2 Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

3 Department of Occupational and Environmental Health, School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei, China

4 School of Nursing, School of Public Health, Yangzhou University, Yangzhou, Jiangsu, China

5 Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

6 Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA

7 Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001S. Lincoln, Urbana, IL, USA

8 Department of Physics and Astronomy, Wayne State University, Detroit, MI, USA

9 Université Grenoble Alpes, INSERM U1209, CNRS UMR 5309, Institut pour l'Avancée des Biosciences (IAB), Team Physiology and Pathophysiology of Sperm cells, 38000 Grenoble, France

10 Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA

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