Autophagy is a highly conserved cytoplasmic degradation pathway that has an impact on many physiological and disease states, including immunity, tumorigenesis and neurodegeneration. Recent studies suggest that autophagy may also have important functions in embryogenesis and development. Many autophagy gene-knockout mice have embryonic lethality at different stages of development. Furthermore, interactions of autophagy with crucial developmental pathways such as Wnt, Shh (Sonic Hedgehog), TGFβ (transforming growth factor β) and FGF (fibroblast growth factor) have been reported. This suggests that autophagy may regulate cell fate decisions, such as differentiation and proliferation. In the present article, we discuss how mammalian autophagy may affect phenotypes associated with development.

Introduction

Autophagy

Autophagy is an evolutionarily conserved pathway that delivers cytoplasmic substrates, such as damaged organelles and cytoplasmic proteins, to lysosomes for degradation. Three classes of autophagy have been identified in mammals, i.e. CMA (chaperone-mediated autophagy), microautophagy, and macroautophagy, that are distinguished chiefly by the mode of cargo delivery to the lysosome. The present review focuses on macroautophagy (hereinafter referred to as autophagy), which has been the most studied overall [1].

Autophagosomes are derived from intracytoplasmic double-membraned cup-shaped structures, called phagophores. The edges of the phagophore extend and seal to form the autophagosome, which sequesters proteins or organelles away from the cytosol. During maturation, the autophagosomes fuse with lysosomes to become acidic autophagolysosomes, where the autophagic substrates are degraded and recycled back to the cytosol in the form of amino acids or macromolecules [2].

Phagophore formation requires PtdIns3P, which is generated by the class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting 34), whose activity for autophagy is enabled by its incorporation in a large macromolecular complex along with Beclin-1 (mammalian Atg6), Atg14 and Vps15 (p150) [3]. The activity of this complex is dependent on upstream autophagy regulators, including ULK1 and ULK2 (mammalian Atg1 orthologues), Atg13 and FIP200 [FAK (focal adhesion kinase)-family interacting protein of 200 kDa] [3].

The biogenesis of mammalian (and yeast) autophagosomes also involves two ubiquitin-like molecules, Atg12 and LC3 (light chain 3)/Atg8 [2,4]. In the first of these reactions, the C-terminal glycine residue of Atg12 is conjugated to ATG5. This is a ubiquitin-like conjugation involving Atg7 as the E1-like enzyme and Atg10 as the E2-like enzyme. The Atg12–Atg5 conjugate then forms an 800 kDa complex with Atg16L1 (Atg16-like 1). Atg16L1 localizes to the outer surface of the pre-autophagosomal structures, and dissociates from fully formed (mature) autophagosomes. Thus Atg16L1-positive vesicles represent pre-autophagosomal structures. In the second ubiquitin-like reaction, LC3 is conjugated to PE (phosphatidylethanolamine) to form lipidated LC3-II by Atg7 and Atg3 as the relevant E1- and E2-like enzymes respectively. LC3-II is specifically targeted to elongating pre-autophagosomal structures and then remains on mature autophagosomes, until after fusion with lysosomes.

Autophagic activity affects the pathogenesis of diverse diseases, such as cancer, neurodegenerative and infectious diseases [5], as it degrades toxic intracytoplasmic contents (such as aggregate-prone proteins and various bacteria), but also serves as a buffer against cellular starvation by generating amino acids and other macromolecules. Studies have suggested roles for autophagy in development, as deletion of various genes that control autophagy results in overt phenotypes or lethality in mice [6]. In some cases, the lethality starts already from the four-cell stage, but knockout of different autophagy genes can cause lethality at later embryonic and even neonatal stages. The major causes are defects in the formation of embryonic structures and organs, cell death, and reduction in neurogenesis and neuronal differentiation [710].

Embryogenesis and development

Embryogenesis starts with the division of one single cell into two-, four- and then eight-cell stages. At E (embryonic day) 2.5, the early morula stage, each cell already possesses distinct apical–basal polarity. The blastocyst containing differentiated cell types is generated at E3.5. The anterio–posterior axis is formed at E6.5, at the same time as the development of the primitive streak [11], followed by the formation of the neural tube at E7.5 [12]. Shh (Sonic Hedgehog), TGFβ (transforming growth factor β) and Wnt family proteins are important factors directing the initiation and closure of the neural tube. In addition, Shh, Wnt and FGF (fibroblast growth factor) are involved in limb formation and organogenesis in later stages of development [1214]. All four signalling molecule families are considered to be morphogens, crucial for patterning and organization in embryonic development [13]. Apart from the morphogen functions, these pathways control proliferation and differentiation of cells throughout and after development.

Role of autophagic proteins in embryogenesis

Knockout of many autophagy-related genes in mice affects early developmental stages, neonatal development and neuronal differentiation (Table 1).

Table 1
Role of autophagy in different stages of embryogenesis
Developmental stage Key steps in development Genes knocked out Phenotype Role of autophagy Developmental pathways active 
Four- to eight-cell Undifferentiated cells, apical basal polarity [11Atg5 Lethal, defects in retina and lung [24Corpse clearance [24Nanog, Oct3/4 [11
E7.5–E16.5 Formation of neural tube, organogenesis [12,14Beclin-1, Ambra1, FIP200 Lethal, massive cell death, loss of amniotic fold [7]; defects in neural tube [8]; increased apoptosis, heart failure liver defects [30Corpse clearance [24Wnt, FGF, Shh, TGFβ [12,14
Postnatal Cell differentiation, proliferation [22Atg7 Abnormal swellings and dystropy of axon [20]; defects in motor fuction [21Local membrane trafficking and turnover [20Wnt, FGF, TGFβ [42,44,45,49
Postnatal Neurogenesis [22Ulk1/Atg5/Atg7 Impairment in axon outgrowth differentiation [22]; swelling of axon, defects in motor function Unknown Wnt, FGF, Shh [53
Developmental stage Key steps in development Genes knocked out Phenotype Role of autophagy Developmental pathways active 
Four- to eight-cell Undifferentiated cells, apical basal polarity [11Atg5 Lethal, defects in retina and lung [24Corpse clearance [24Nanog, Oct3/4 [11
E7.5–E16.5 Formation of neural tube, organogenesis [12,14Beclin-1, Ambra1, FIP200 Lethal, massive cell death, loss of amniotic fold [7]; defects in neural tube [8]; increased apoptosis, heart failure liver defects [30Corpse clearance [24Wnt, FGF, Shh, TGFβ [12,14
Postnatal Cell differentiation, proliferation [22Atg7 Abnormal swellings and dystropy of axon [20]; defects in motor fuction [21Local membrane trafficking and turnover [20Wnt, FGF, TGFβ [42,44,45,49
Postnatal Neurogenesis [22Ulk1/Atg5/Atg7 Impairment in axon outgrowth differentiation [22]; swelling of axon, defects in motor function Unknown Wnt, FGF, Shh [53

Autophagy is important during critical mammalian developmental stages in which nutrients are restricted. One such stage is pre-implantation development of embryos [15]. Autophagic activity is low in unfertilized oocytes and increases shortly after fertilization. It is transiently suppressed between the late one-cell and middle two-cell stages and is then activated again after the late two-cell stage. Autophagosome formation rates continue to increase through the four- to eight-cell phase. The complete loss of autophagy in oocytes, as seen in Atg5-knockout mice under the zona pellucida glycoprotein (Zp3) oocyte-specific promoter halted proper development of embryos before the blastocyst phase. Atg5-deficient oocytes fertilized by Atg5-null sperm failed to develop beyond the four- and eight-cell stages, but could develop if fertilized by wild-type sperm. A lack of autophagy from the start of oogenesis does not seem to affect oocyte formation or fertilization. It is not known whether the main role of autophagy in pre-implantation development is to provide nutrients to the growing embryo or to clear maternal proteins [15,16]. Other possible consequences of autophagy deficiency are also possible in this context, including secondary effects on the ubiquitin–proteasome system, which may have an impact on the co-ordination of the levels of critical proteins required for regulating cell division. It is possible that some of these secondary consequences of autophagy compromise may contribute to the necessity for autophagy during T-cell development. Deletion of Atg5 or Atg7 in T-cells was accompanied by a decrease in thymocyte and peripheral T-cell numbers and also resulted in a decrease in T-cell survival [17].

Sudden termination of the fetal nutrient supply from the mother presents a stressful situation for the newborn infant before it establishes breastfeeding. During this transition period, autophagy provides necessary nutrients to the infant through increased turnover of proteins. This has been shown experimentally in a transgenic mouse model expressing GFP-tagged LC3 to visualize autophagosomes in vivo [18]. In these mice, autophagy in various tissues increases soon after birth, peaks at ~6 h after birth, and declines back to basal levels within 24–48 h [18]. To assess the specific role of autophagy, the effect of starvation during this critical period in Atg5- and Atg7-knockout mice was studied. Whereas mice generated from animals with conditional deletion of Atg5 in oocytes and Atg5-null sperm cannot develop beyond the four- and eight-cell stages, conventional Atg5−/− mice generated by mating Atg5+/− mice survive until after birth, since the maternally derived Atg5 proteins stored in oocytes is sufficient to rescue the autophagy-deficient phenotype of early embryogenesis [15,16]. These mice develop normally with only a slightly lower birthweight for the Atg5−/− mice, and a significant weight reduction in the Atg7−/− mice [18,19]. Atg5- and Atg7-knockout mice die within 1 day of birth [18,19]. Under forced starvation conditions, the survival time of the Atg5-knockout mice was nearly half (12–13 h) that of their Atg5+/− or wild-type littermates (21–24 h) [18]. These experiments support the role of autophagy in normal developmental processes that occur in the context of diminished nutrient supply, lending further importance to the need for the functional turnover of amino acids. However, it is possible that other mechanisms may also be relevant. A recent study found that deletion of Chk2 (checkpoint kinase 2) rescues postnatal lethality in Atg7−/− mice, suggesting that Atg7 may regulate p53-dependent cell cycle and cell death pathways during metabolic stress [4]. On the other hand, the ablation of Atg7 leads to abnormal swellings and dystrophy of Purkinje cell axon terminals in the deep cerebellar nuclei [20], and mice lacking Atg7 in neuronal cells develop progressive deficits in motor function leading to the accumulation of ubiquitin-containing inclusion bodies [21]. Thus neuronal abnormalities may also contribute to the neonatal lethality of these mice.

The idea that autophagy is important for neuronal development is also supported by observations that inactivation of Ulk1 (Atg1) in immature granule cells impairs axon outgrowth and differentiation of neurons. Ulk1 activity at the growth cone is thus crucial for the formation of fibres, and allows the progression of cerebellar development [22]. In addition, Ulk1−/− mice show increased reticulocyte numbers with delayed mitochondrial clearance, suggesting that ULK1 is involved in mitophagy in reticulocyte development [23].

Beclin-1-knockout mice have early embryonic lethality (compared with Atg5- or Atg7-null mice derived from hemizygous parents which are live-born, but die soon after birth) [7]. Beclin-1-knockout mice have an early delay in development, so that, at E7.5, the embryo size is already severely reduced and massive cell death is observed [7]. Beclin-1 is expressed strongly in the extraembryonic visceral endoderm in wild-type animals. In null embryos, the structures are formed, despite severe growth delay. Mutant embryos show a loss of amniotic fold and no closure of the pro-amniotic canal [7]. In vitro, Beclin-1−/− embryonic stem cells fail to form expanded cystic embryoid bodies, which are mimics of the blastocyst. This is found to be due to decreased cell clearance at the core of the embryonic body [7]. The decrease in apoptotic corpse clearance appears to result from the failure to display the PS (phosphatidylserine) ‘eat me’ signal normally expressed on apoptotic cell surfaces in the Beclin-1−/− mutants. The release of the LPC (lysophosphatidylcholine) ‘come and get me’ signal, is also reduced. (LPC normally functions as a chemoattractant for phagocyte recruitment.) The cause for the decrease might be a consequence of ATP generation by autophagy. Autophagy releases a number of molecules which enter the ATP production cycle. ATP levels were lower in Beclin-1−/− embryoid bodies. Since ATP is crucial for the generation of apoptotic signals, autophagy might be important for apoptotic corpse clearance by producing ATP for apoptotic signal expression [24]. It is also possible that some of the phenotypes in the Beclin-1-null embryos may be autophagy-independent due to reduced p53 levels, as Beclin-1 indirectly regulates p53 degradation [25].

Ambra1 (activating molecule in Beclin-1-regulated autophagy 1) is part of the autophagy induction machinery and is required for full Beclin-1 activation and enhances the Beclin-1–Vps34 interaction [8]. Ambra1 is unique to vertebrates and is highly conserved among different species. It plays a major role in embryonic development [8]. Furthermore, it is involved in organogenesis in zebrafish and neurogenesis in NSCs (neural stem cells) [26]. During early embryogenesis, its expression is mostly restricted to the neuroepithelium. Ambra1−/− mutants have embryonic lethality at E16.5, with defects in the neural tube and hyperproliferation, followed by massive apoptotic cell death [8]. The neural plate defects result in severe abnormalities, such as exencephaly and spina bifida. So autophagy might be important for tissue-specific developmental regulation [8], although some of these Ambra1-knockout phenotypes may also be autophagy-independent.

FIP200, a 200 kDa protein with a large coiled-coil region, which is an inhibitor of FAK [27,28], is conserved from Drosophila to humans [28]. It regulates many cellular processes, including cell migration, proliferation, cell size and apoptosis, due to its multiple interaction partners, such as FAK, Pyk2 (proline-rich tyrosine kinase 2), TSC1 (tuberous sclerosis complex 1), p53, ASK1 (apoptosis signal-regulating kinase 1) and TRAF (tumour-necrosis-factor-receptor-associated factor) [29]. Apart from these partners, FIP200 interacts with ULK1 to modulate autophagosome formation. FIP200-deficient cells are not able to form autophagosomes [29]. FIP200 is essential for embryonic development. Knockout mice have embryonic lethality between E13.5 and E16.5 because of heart failure and liver defects [30]. This is mainly due to increased apoptosis, which leads to decreased cell numbers and thus thin ventricular walls and liver lesions. Some of these effects may be independent of autophagy, as FIP200 interacts with ASK1 and TRAF2, which can modulate JNK (c-Jun N-terminal kinase) activation in response to TNFα (tumour necrosis factor α)-induced apoptotic signals [30]. In addition, FIP200 is crucial for HSC (haemopoietic stem cell) maintenance in embryonic development. FIP200 deletion increases the rate of HSC proliferation, mitochondrial mass and ROS (reactive oxygen species). Similar effects were found in NSCs.

FIP200 deletion results in loss of NSCs and reduction in neurogenesis in postnatal brain (subventricular zone and dentate gyrus). This effect can be reversed by treating with antioxidants [31].

Interaction of developmental pathways and autophagy

Embryogenesis and neuronal development is controlled by central signalling pathways, such as Shh, TGFβ and Wnt. In embryogenesis, these pathways are essential for patterning, formation of the body axis, limb development and formation of the neural tube [1214,32]. Furthermore, each pathway regulates individual cell fate in embryonic and adult tissue. It has been reported that autophagy interacts with these developmental pathways and FGF signalling (Figure 1). The cross-talk is crucial for cell fate decisions, where autophagy has pro-differentiation and pro-proliferation roles, as well as a role in cell cycle arrest and apoptosis [3336]. In addition, the interaction is a likely cause for the defects in development observed in the knockout mice, since the pathways are important for formation of embryonic structures.

Interaction of autophagy and developmental pathways: TGFβ activates autophagy via Smad to cause cell cycle arrest/apoptosis

Figure 1
Interaction of autophagy and developmental pathways: TGFβ activates autophagy via Smad to cause cell cycle arrest/apoptosis

Shh inhibits autophagy via Patched. FGF can inhibit autophagy through the mTOR pathway to block differentiation, but can in turn be inhibited by autophagy. Autophagy inhibits Wnt by degrading Dvl, in addition GSK3β, an important factor in the Wnt pathway, can activate autophagy.

Figure 1
Interaction of autophagy and developmental pathways: TGFβ activates autophagy via Smad to cause cell cycle arrest/apoptosis

Shh inhibits autophagy via Patched. FGF can inhibit autophagy through the mTOR pathway to block differentiation, but can in turn be inhibited by autophagy. Autophagy inhibits Wnt by degrading Dvl, in addition GSK3β, an important factor in the Wnt pathway, can activate autophagy.

The Shh signalling pathway plays an important role in patterning of the central nervous system, neural tube development and limb development. Previously, we reported that the Hedgehog signalling pathway inhibits autophagosome synthesis, both in basal and in autophagy-induced conditions [33,37]. On the other hand, Shh induces autophagy in hippocampal neurons [38]. This might be due to dual functions of Shh serving as mitogen in NSCs, but as an autophagy enhancer in differentiated neurons [37,3941].

TGFβ signalling is one of the most important players that drives developmental programmes that control cell behaviour, and plays crucial roles in pluripotency and differentiation of embryonic stem cells in vitro [42]. Recent evidence demonstrates that TGFβ activates autophagy through Smad in hepatocellular carcinoma cells and mammary carcinoma cells, and induces cell cycle arrest and inhibits cell growth [43]. It also up-regulates several autophagy-related genes, such as Beclin-1, Atg5, Atg7 and DAPK (death-associated protein kinase) by Smads and JNK [35].

The Wnt signalling pathway is crucial for embryogenesis, planar cell polarity and maintenance of stem cell fate [44,45]. The canonical pathway is activated by binding of the ligand Wnt to the transmembrane receptor Frizzled, which results in activation of Dvl (Dishevelled). Dvl, in turn, inhibits GSK3β (glycogen synthase kinase 3β), a factor in the β-catenin-inhibiting complex. Thus β-catenin is released and translocates into the nucleus for activation of target genes [46,47]. Autophagy was found to negatively regulate Wnt signalling by degrading Dvl. The von Hippel–Lindau protein ubiquitinates Dvl and marks it for autophagy. In this context, a loss of autophagy was reported to correlate with an increase in colon tumours [34]. On the other hand, GSK3β (an inhibitor of Wnt signalling) was reported to stimulate autophagy through an mTOR (mammalian target of rapamycin)-dependent mechanism [48]. There may be a feedback mechanism through the master sensor GSK3β, which regulates Wnt signalling by multiple mechanisms.

FGF signalling has wide effects on many downstream pathways involving cell proliferation, survival, differentiation and migration [49]. In embryogenesis, it has a crucial role in limb development and mesoderm induction. FGF can activate Ras and MAPK (mitogen-activated protein kinase) kinase pathways leading to proliferation of PI3K/Akt resulting in survival [50]. It also activates calcium signalling [50]. Autophagy interacts with FGF in multiple cell types (e.g. cardiomyocytes, chondrocytes and adipose tissue) to regulate crucial developmental and signalling steps [36,51,52]. In the heart, FGF inhibits autophagy to suppress cardiac cell differentiation. It prevents premature differentiation of progenitor cells [36]. However, it remains unclear how autophagy promotes differentiation in this context. Autophagy deficiency in adipose tissue leads to mitochondrial dysfunction and subsequent increase in FGF21 production, which, in turn, results in diet-induced obesity and insulin resistance [52]. This indicates a feedback loop between FGF and autophagy. The cross-talk of the two pathways is crucial in developmental turning points.

Conclusion

The analyses of various autophagy-knockout mice provided the first evidence that autophagy might have multiple functions during development. Most mechanisms that have been proposed have considered conventional functions of autophagy, where autophagy is responsible for clearance, e.g. mitochondrial or corpse clearance, which, in turn, frees nutrients for amino acid supply. In some cases, it may be important to consider roles for specific proteins that are independent of autophagy, e.g. in the case of FIP200 and its interactions with ASK1 and TRAF2. For many of the phenotypes, however, the mechanism remains unknown. Since embryogenesis is mainly governed by developmental pathways, such as Shh, TGFβ, Wnt and FGF, and there is an intensive cross-talk of autophagic proteins with these pathways (with implications in differentiation and proliferation of a variety of cell types), autophagy might regulate embryogenesis through these routes. However, it is likely that there are further mechanisms and regulatory loops to be discovered.

5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders: A joint Biochemical Society/European Society for Neurochemistry Focused Meeting held at the University of Bath, U.K., 23–26 June 2013. Organized and Edited by Marcus Rattray (University of Bradford, U.K.) and Rob Williams (University of Bath, U.K.).

Abbreviations

     
  • Ambra1

    activating molecule in Beclin-1-regulated autophagy 1

  •  
  • ASK1

    apoptosis signal-regulating kinase 1

  •  
  • Atg16L1

    Atg16-like 1

  •  
  • Dvl

    Dishevelled

  •  
  • E

    embryonic day

  •  
  • FAK

    focal adhesion kinase

  •  
  • FGF

    fibroblast growth factor

  •  
  • FIP200

    FAK-family interacting protein of 200 kDa

  •  
  • GSK3β

    glycogen synthase kinase 3β

  •  
  • HSC

    haemopoietic stem cell

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LC3

    light chain 3

  •  
  • LPC

    lysophosphatidylcholine

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NSC

    neural stem cell

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • Shh

    Sonic Hedgehog

  •  
  • TGFβ

    transforming growth factor β

  •  
  • TRAF

    tumour-necrosis-factor-receptor-associated factor

  •  
  • Vps

    vacuolar protein sorting

Funding

We are grateful for a Wellcome Trust Principal Fellowship (to D.C.R.), Friedrich-Ebert-Stiftung (to X.W.) and Korea–UK Alzheimer's Disease Research consortium programme from the Ministry of Health and Welfare, Republic of Korea (to H.W.).

References

References
1
Jimenez-Sanchez
M.
Thomson
F.
Zavodszky
E.
Rubinsztein
D.C.
Autophagy and polyglutamine diseases
Prog. Neurobiol.
2012
, vol. 
97
 (pg. 
67
-
82
)
2
Rubinsztein
D.C.
Codogno
P.
Levine
B.
Autophagy modulation as a potential therapeutic target for diverse diseases
Nat. Rev. Drug Discovery
2012
, vol. 
11
 (pg. 
709
-
730
)
3
Weidberg
H.
Shvets
E.
Elazar
Z.
Biogenesis and cargo selectivity of autophagosomes
Annu. Rev. Biochem.
2011
, vol. 
80
 (pg. 
125
-
156
)
4
Lee
I.H.
Kawai
Y.
Fergusson
M.M.
Rovira
I.I.
Bishop
A.J.
Motoyama
N.
Cao
L.
Finkel
T.
Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress
Science
2012
, vol. 
336
 (pg. 
225
-
228
)
5
Choi
A.M.
Ryter
S.W.
Levine
B.
Autophagy in human health and disease
N. Engl. J. Med.
2013
, vol. 
368
 (pg. 
1845
-
1846
)
6
Cecconi
F.
Levine
B.
The role of autophagy in mammalian development: cell makeover rather than cell death
Dev. Cell
2008
, vol. 
15
 (pg. 
344
-
357
)
7
Yue
Z.
Jin
S.
Yang
C.
Levine
A.J.
Heintz
N.
Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor
Proc. Natl. Acad. Sci. U.S.A.
2003
, vol. 
100
 (pg. 
15077
-
15082
)
8
Fimia
G.M.
Stoykova
A.
Romagnoli
A.
Giunta
L.
Di Bartolomeo
S.
Nardacci
R.
Corazzari
M.
Fuoco
C.
Ucar
A.
Schwartz
P.
, et al. 
Ambra1 regulates autophagy and development of the nervous system
Nature
2007
, vol. 
447
 (pg. 
1121
-
1125
)
9
Zhang
J.
Randall
M.S.
Loyd
M.R.
Dorsey
F.C.
Kundu
M.
Cleveland
J.L.
Ney
P.A.
Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation
Blood
2009
, vol. 
114
 (pg. 
157
-
164
)
10
Mizushima
N.
Levine
B.
Autophagy in mammalian development and differentiation
Nat. Cell Biol.
2010
, vol. 
12
 (pg. 
823
-
830
)
11
Takaoka
K.
Hamada
H.
Cell fate decisions and axis determination in the early mouse embryo
Development
2012
, vol. 
139
 (pg. 
3
-
14
)
12
Copp
A.J.
Greene
N.D.
Murdoch
J.N.
The genetic basis of mammalian neurulation
Nat. Rev. Genet.
2003
, vol. 
4
 (pg. 
784
-
793
)
13
Tabata
T.
Genetics of morphogen gradients, Nat
Rev. Genet.
2001
, vol. 
2
 (pg. 
620
-
630
)
14
Harvey
R.P.
Patterning the vertebrate heart
Nat. Rev. Genet.
2002
, vol. 
3
 (pg. 
544
-
556
)
15
Tsukamoto
S.
Kuma
A.
Murakami
M.
Kishi
C.
Yamamoto
A.
Mizushima
N.
Autophagy is essential for preimplantation development of mouse embryos
Science
2008
, vol. 
321
 (pg. 
117
-
120
)
16
Tsukamoto
S.
Kuma
A.
Mizushima
N.
The role of autophagy during the oocyte-to-embryo transition
Autophagy
2008
, vol. 
4
 (pg. 
1076
-
1078
)
17
Stephenson
L.M.
Miller
B.C.
Ng
A.
Eisenberg
J.
Zhao
Z.
Cadwell
K.
Graham
D.B.
Mizushima
N.N.
Xavier
R.
Virgin
H.W.
Swat
W.
Identification of Atg5-dependent transcriptional changes and increases in mitochondrial mass in Atg5-deficient T lymphocytes
Autophagy
2009
, vol. 
5
 (pg. 
625
-
635
)
18
Ravikumar
B.
Vacher
C.
Berger
Z.
Davies
J.E.
Luo
S.
Oroz
L.G.
Scaravilli
F.
Easton
D.F.
Duden
R.
O’Kane
C.J.
Rubinsztein
D.C.
Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease
Nat. Genet.
2004
, vol. 
36
 (pg. 
585
-
595
)
19
Komatsu
M.
Waguri
S.
Ueno
T.
Iwata
J.
Murata
S.
Tanida
I.
Ezaki
J.
Mizushima
N.
Ohsumi
Y.
Uchiyama
Y.
, et al. 
Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice
J. Cell Biol.
2005
, vol. 
169
 (pg. 
425
-
434
)
20
Komatsu
M.
Wang
Q.J.
Holstein
G.R.
Friedrich
V.L.
Jr
Iwata
J.
Kominami
E.
Chait
B.T.
Tanaka
K.
Yue
Z.
Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration
Proc. Natl. Acad. Sci. U.S.A.
2007
, vol. 
104
 (pg. 
14489
-
14494
)
21
Komatsu
M.
Waguri
S.
Chiba
T.
Murata
S.
Iwata
J.
Tanida
I.
Ueno
T.
Koike
M.
Uchiyama
Y.
Kominami
E.
Tanaka
K.
Loss of autophagy in the central nervous system causes neurodegeneration in mice
Nature
2006
, vol. 
441
 (pg. 
880
-
884
)
22
Di Bartolomeo
S.
Nazio
F.
Cecconi
F.
The role of autophagy during development in higher eukaryotes
Traffic
2010
, vol. 
11
 (pg. 
1280
-
1289
)
23
Kundu
M.
Lindsten
T.
Yang
C.Y.
Wu
J.
Zhao
F.
Zhang
J.
Selak
M.A.
Ney
P.A.
Thompson
C.B.
Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation
Blood
2008
, vol. 
112
 (pg. 
1493
-
1502
)
24
Qu
X.
Zou
Z.
Sun
Q.
Luby-Phelps
K.
Cheng
P.
Hogan
R.N.
Gilpin
C.
Levine
B.
Autophagy gene-dependent clearance of apoptotic cells during embryonic development
Cell
2007
, vol. 
128
 (pg. 
931
-
946
)
25
Liu
J.
Xia
H.
Kim
M.
Xu
L.
Li
Y.
Zhang
L.
Cai
Y.
Norberg
H.V.
Zhang
T.
Furuya
T.
, et al. 
Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13
Cell
2011
, vol. 
147
 (pg. 
223
-
234
)
26
Cecconi
F.
Di Bartolomeo
S.
Nardacci
R.
Fuoco
C.
Corazzari
M.
Giunta
L.
Romagnoli
A.
Stoykova
A.
Chowdhury
K.
Fimia
G.M.
Piacentini
M.
A novel role for autophagy in neurodevelopment
Autophagy
2007
, vol. 
3
 (pg. 
506
-
508
)
27
Abbi
S.
Ueda
H.
Zheng
C.
Cooper
L.A.
Zhao
J.
Christopher
R.
Guan
J.L.
Regulation of focal adhesion kinase by a novel protein inhibitor FIP200
Mol. Biol. Cell
2002
, vol. 
13
 (pg. 
3178
-
3191
)
28
Gan
B.
Melkoumian
Z.K.
Wu
X.
Guan
K.L.
Guan
J.L.
Identification of FIP200 interaction with the TSC1–TSC2 complex and its role in regulation of cell size control
J. Cell Biol.
2005
, vol. 
170
 (pg. 
379
-
389
)
29
Hara
T.
Takamura
A.
Kishi
C.
Iemura
S.
Natsume
T.
Guan
J.L.
Mizushima
N.
FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells
J. Cell Biol.
2008
, vol. 
181
 (pg. 
497
-
510
)
30
Gan
B.
Peng
X.
Nagy
T.
Alcaraz
A.
Gu
H.
Guan
J.L.
Role of FIP200 in cardiac and liver development and its regulation of TNFα and TSC–mTOR signaling pathways
J. Cell Biol.
2006
, vol. 
175
 (pg. 
121
-
133
)
31
Wang
C.
Liang
C.C.
Bian
Z.C.
Zhu
Y.
Guan
J.L.
FIP200 is required for maintenance and differentiation of postnatal neural stem cells
Nat. Neurosci.
2013
, vol. 
16
 (pg. 
532
-
542
)
32
Raya
A.
Izpisua Belmonte
J.C.
Left–right asymmetry in the vertebrate embryo: from early information to higher-level integration
Nat. Rev. Genet.
2006
, vol. 
7
 (pg. 
283
-
293
)
33
Jimenez-Sanchez
M.
Menzies
F.M.
Chang
Y.Y.
Simecek
N.
Neufeld
T.P.
Rubinsztein
D.C.
The Hedgehog signalling pathway regulates autophagy
Nat. Commun.
2012
, vol. 
3
 pg. 
1200
 
34
Gao
C.
Cao
W.
Bao
L.
Zuo
W.
Xie
G.
Cai
T.
Fu
W.
Zhang
J.
Wu
W.
Zhang
X.
Chen
Y.G.
Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation
Nat. Cell Biol.
2010
, vol. 
12
 (pg. 
781
-
790
)
35
Suzuki
H.I.
Kiyono
K.
Miyazono
K.
Regulation of autophagy by transforming growth factor-β (TGF-β) signaling
Autophagy
2010
, vol. 
6
 (pg. 
645
-
647
)
36
Zhang
J.
Liu
J.
Liu
L.
McKeehan
W.L.
Wang
F.
The fibroblast growth factor signaling axis controls cardiac stem cell differentiation through regulating autophagy
Autophagy
2012
, vol. 
8
 (pg. 
690
-
691
)
37
Han
Y.G.
Spassky
N.
Romaguera-Ros
M.
Garcia-Verdugo
J.M.
Aguilar
A.
Schneider-Maunoury
S.
Alvarez-Buylla
A.
Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells
Nat. Neurosci.
2008
, vol. 
11
 (pg. 
277
-
284
)
38
Petralia
R.S.
Schwartz
C.M.
Wang
Y.X.
Kawamoto
E.M.
Mattson
M.P.
Yao
P.J.
Sonic hedgehog promotes autophagy in hippocampal neurons
Biol. Open
2013
, vol. 
2
 (pg. 
499
-
504
)
39
Lai
K.
Kaspar
B.K.
Gage
F.H.
Schaffer
D.V.
Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo
Nat. Neurosci.
2003
, vol. 
6
 (pg. 
21
-
27
)
40
Palma
V.
Lim
D.A.
Dahmane
N.
Sanchez
P.
Brionne
T.C.
Herzberg
C.D.
Gitton
Y.
Carleton
A.
Alvarez-Buylla
A.
Ruiz i Altaba
A.
Sonic hedgehog controls stem cell behavior in the postnatal and adult brain
Development
2005
, vol. 
132
 (pg. 
335
-
344
)
41
Breunig
J.J.
Sarkisian
M.R.
Arellano
J.I.
Morozov
Y.M.
Ayoub
A.E.
Sojitra
S.
Wang
B.
Flavell
R.A.
Rakic
P.
Town
T.
Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling
Proc. Natl. Acad. Sci. U.S.A.
2008
, vol. 
105
 (pg. 
13127
-
13132
)
42
Massague
J.
TGFβ signalling in context
Nat. Rev. Mol. Cell Biol.
2012
, vol. 
13
 (pg. 
616
-
630
)
43
Kiyono
K.
Suzuki
H.I.
Matsuyama
H.
Morishita
Y.
Komuro
A.
Kano
M.R.
Sugimoto
K.
Miyazono
K.
Autophagy is activated by TGF-β and potentiates TGF-β-mediated growth inhibition in human hepatocellular carcinoma cells
Cancer Res.
2009
, vol. 
69
 (pg. 
8844
-
8852
)
44
MacDonald
B.T.
Tamai
K.
He
X.
Wnt/β-catenin signaling: components, mechanisms, and diseases
Dev. Cell
2009
, vol. 
17
 (pg. 
9
-
26
)
45
van Amerongen
R.
Nusse
R.
Towards an integrated view of Wnt signaling in development
Development
2009
, vol. 
136
 (pg. 
3205
-
3214
)
46
Rao
T.P.
Kuhl
M.
An updated overview on Wnt signaling pathways: a prelude for more
Circ. Res.
2010
, vol. 
106
 (pg. 
1798
-
1806
)
47
Backues
S.K.
Klionsky
D.J.
Autophagy gets in on the regulatory act
J. Mol. Cell Biol.
2011
, vol. 
3
 (pg. 
76
-
77
)
48
Zhai
P.
Sadoshima
J.
Glycogen synthase kinase-3β controls autophagy during myocardial ischemia and reperfusion
Autophagy
2012
, vol. 
8
 (pg. 
138
-
139
)
49
Turner
N.
Grose
R.
Fibroblast growth factor signalling: from development to cancer
Nat. Rev. Cancer
2010
, vol. 
10
 (pg. 
116
-
129
)
50
Iwata
T.
Hevner
R.F.
Fibroblast growth factor signaling in development of the cerebral cortex
Dev., Growth Differ.
2009
, vol. 
51
 (pg. 
299
-
323
)
51
Kim
K.H.
Jeong
Y.T.
Oh
H.
Kim
S.H.
Cho
J.M.
Kim
Y.N.
Kim
S.S.
Kim do
H.
Hur
K.Y.
Kim
H.K.
, et al. 
Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine
Nat. Med.
2013
, vol. 
19
 (pg. 
83
-
92
)
52
Settembre
C.
Arteaga-Solis
E.
McKee
M.D.
de Pablo
R.
Al Awqati
Q.
Ballabio
A.
Karsenty
G.
Proteoglycan desulfation determines the efficiency of chondrocyte autophagy and the extent of FGF signaling during endochondral ossification
Genes Dev.
2008
, vol. 
22
 (pg. 
2645
-
2650
)
53
Hagemann
A.I.
Scholpp
S.
The tale of the three brothers: Shh, Wnt, and Fgf during development of the thalamus
Front. Neurosci.
2012
, vol. 
6
 pg. 
76
 

Author notes

1

These authors contributed equally to this paper.