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Interpreting Neonatal Lethal Phenotypes in Mouse Mutants: Insights Into Gene Function and Human Diseases

Institut de Recherche en Immunologie et Cancérologie and Departments of Pharmacology and Molecular Biology, Université de Montréal, Montreal, Quebec, Canada

ABSTRACT

I. INTRODUCTION

II. SURVIVING PARTURITION

A. Morphological Defects

1. Gross appearance

2. Neural tube defects

3. Omphalocele

B. Hemorrhage

1. Vasculogenesis defects

2. Hemostasis defects

III. THE FIRST BREATH OF AIR

A. Lung Defects

1. Branching morphogenesis

2. Lung maturation

3. Transition to air breathing

4. Upper airways

B. Cardiovascular Defects

1. Outflow tract and aortic arch

2. Ductus arteriosus

C. Neuromuscular Defects

1. Nervous system

2. Neuromuscular junction

3. Respiratory muscles

D. Skeletal Defects

1. Rib cage

2. Vertebrae

IV. ABNORMAL FEEDING

A. Neuromuscular Defects

1. Olfaction

2. Sensory-motor

3. Hypothalamus

4. Muscles

B. Craniofacial Defects

C. Parental Behavior

V. HOMEOSTASIS DEFAULTS

A. Hypoglycemia

B. Transepidermal Water Loss

C. Renal Failure

VI. CONCLUSIONS

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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In the last 25 years, we have witnessed the enormous power of gene targeting for dissecting out the physiological functions of mammalian genes. Nearly 4,000 knockout mouse models have been generated to date (24). High-throughput approaches to generate knockout mice have been developed (158), suggesting that generating functional mutations in every gene of this mammalian genome is achievable. It is expected that in a few years only, over 70% of the mouse genome could be mutated, with the possible production of over 20,000 mutant lines of mice (see Refs. 24 and 56 for relevant web resources). Although gene-targeting techniques are straightforward and accessible to most laboratories, the largest investment of time and money in these projects is not in generating mutant mice but rather in their analysis. Indeed, unless a mutation causes a morphologically obvious phenotype, the analysis of complex phenotypes can be very challenging. This is especially true for mutants dying perinatally, which represent a substantial proportion of lethal phenotypes in the mouse (Fig. 1). In the absence of overt defects, such as organ agenesis for example, death itself becomes the starting point for phenotypic investigation in this class of mutants.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Timing of death in mice harboring lethal mutations. Calculations were made from the number of genotypes associated with lethality obtained from the Phenotype Ontology Database resource (www.informatics.jax.org).

View larger version (33K): [in this window] [in a new window]   FIG. 2. Neonatal death in mutant mice reveals complex biological networks. Cause-to-effect relationships between organ defects and the affected physiological processes critical for neonatal survival are shown.

View larger version (8K): [in this window] [in a new window]   FIG. 3. Twenty-four hours to survive birth in mouse mutants. Top: predicted survival curves when essential physiological processes are impaired in newborn mice (assuming a fully penetrant phenotype). Bottom: specific examples of gene mutations and associated physiological defects that compromise neonatal survival. The gray lines represent the window of neonatal lethality.

	II. SURVIVING PARTURITION

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The first life-threatening event associated with neonatal survival is parturition itself. Although the eutherian mammalian fetus is evolutionarily adapted to sustain parturition, the stress encountered by the pups at parturition is high enough to cause lethal damage to abnormal pups. The aim of this section is not to come up with a list of all neonatal lethal mutants presenting catastrophic or morphological defects. Instead, we wish to illustrate how analysis of such mutants has allowed deciphering the function of genes involved in important embryonic processes, whose loss of function can be revealed by neonatal death.

A. Morphological Defects

1. Gross appearance

A number of mutations leading to severe morphological (catastrophic) defects have been described in the mouse. Most of these defects do not compromise embryonic survival and are only revealed when the pups are found dead, or not found (cannibalized), after birth. One of the most striking examples of morphological defects is probably the Shh knockout. Some Shh mutant mice reach birth with a morphology very distinct from that of a normal pup, and they are likely eaten immediately after delivery (17). Less severe, still impressive mutants include Wnt5a mutant mice, which are born with a dwarf appearance because of shortened jaws and limbs (173), and Ctnnbip1 null mice, which lack the anterior structure of the head (Fig. 4 A) (138). A total absence of limbs was described in Fgf10 mutants, but in these mice death after birth was due to the absence of lungs (106, 144).

View larger version (79K): [in this window] [in a new window]   FIG. 4. Reaching birth with severe morphological defects. A: exencephaly, absence of eyes, nose, maxillary and rostral skull in Ctnnbip1 knockout mice. [Modified from Satoh et al. (138), copyright 2004 National Academy of Sciences, USA.] B: a severe case of omphalocele (arrow in right panel) observed in Rock1 mutant newborn. Notice the open eyelid in the homozygous mutant (arrowhead). [Modified from Shimizu et al. (146), with permission from the Rockefeller University Press.] C: severe hemorrhage in the head of Itgav mutant neonates. Arrows indicate comparable regions. [Modified from Bader et al. (2), copyright 1998, with permission from Elsevier.]

Morphological defects can lead to the exposure of internal components, such as neural or intestinal tissues. Such defects are problematic for newborn mice for two reasons. First, exposed tissues can be lethally damaged during parturition, leading to birth trauma. Second, the pups can be eaten as they are being cleaned by the mother. A good example is provided by the Cart1 gene knockout. Newborns were found to lack brain tissues and skull after birth. However, analysis of E18.5 fetuses revealed acrania as expected, but the brain was present, indicating that it had been cannibalized during parturition (175). Exposure of neural tissues can be a consequence of abnormal neural tube closure, and mouse mutants represent useful models of neural tube defects, which are relatively common in humans (0.2–3.5 per 2,000 pregnancies). More than 80 mutant mouse models present neural tube defects, and their characterization has helped understanding the molecular pathways involved in neural tube closure (55, 162).

3. Omphalocele

An omphalocele is a normal transient structure occurring during mouse embryonic development and consists of a protrusion of the gut outside the body wall. Failure in resorption of the omphalocele during the fetal phase of development leads to herniation of the midgut and is hazardous for newborn mice survival. Indeed, as described for the Rock1 gene deletion (Fig. 4B), the exposed gut can be easily eaten by the mother, which results in neonatal death (146). Several other gene mutations have been shown to cause some degree of body wall malformations in the mouse. Analysis of these mutant mice has helped to uncover some of the key genes and pathways involved in the morphogenesis, movement, and closure of the ventral body wall (7). Therefore, the mouse represents a unique model to study the genetic basis of body wall closure defects in human, which occur in 1 in 3,000–10,000 live births (104).

Knowing the precise cause of death associated with such catastrophic mutations has low experimental value, since the phenotype itself is informative enough to define the biological function of the mutated gene. However, these examples illustrate how neonatal death in mutant mice can sometimes reveal gene functions that are essential for embryonic development such as those involved in embryonic patterning.

B. Hemorrhage

1. Vasculogenesis defects

Failure in the establishment and maintenance of vascular circulation is an important cause of embryonic lethality in mouse mutants (30). However, certain mutations affecting vasculogenesis are revealed by neonatal death of the mice. Examples include the inactivation of genes involved in platelet-derived growth factor (PDGF)-BB signaling. Disruption of both the gene encoding PDGF-BB (Pdgfb) or its receptor (Pdgfrb) leads to fetal hemorrhage and perinatal death (95, 149). Notably, mutant mice expressing a kinase-deficient PDGF β-receptor also exhibit hemorrhage in multiple tissues at birth (156). These simple examples illustrate how phenocopies of known mutants can be exploited to dissect the components of a signal transduction pathway. As shown in Figure 4C, targeted deletion of the Itgav gene, which encodes the integrin alpha V isoform, is associated with severe intracranial and intestinal hemorrhage at birth (2). This model has provided useful information on the role of integrin alpha V in vascular development in specific tissues. Another example is provided by the loss of function of Cxcr4, a gene important for gastrointestinal vascularization. Deletion of this gene leads to intestinal hemorrhage, and the mutants that reach term and are born cannot survive for more than 1 h (153). Although hemorrhage in the fetus can be responsible for perinatal death, the stress encountered during normal parturition can also lead to the rupture of blood vessels in animals that reach term.

2. Hemostasis defects

Abnormal blood coagulation in mutant mice can also be revealed at birth. Targeted deletion of the Proc gene, which encodes a major anticoagulant, leads to the occlusion of the vasculature and results in fatal thrombosis and neonatal death. Protein C-deficient pups are either born dead or severely bruised on the head with subsequent death. However, pups can survive up to 24 h when delivered by cesarean section (74), a finding which emphasizes that the trauma of birth itself can be responsible for death. Inactivating mutations of coagulation factor genes can also result in abnormal hemostasis and neonatal death. Mice with a disruption of the F5 (factor V) gene die within 2 h of massive intra-abdominal hemorrhage (31), whereas F7 (factor VII) knockout mice survive up to 24 h after birth (135). Mutant mice showing neonatal death due to defects in the procoagulant, anticoagulant, and fibrinolytic pathways represent suitable models of coagulopathies in humans. Their study has brought forth the general concept that hemostasis is regulated in a tissue-specific manner (99).

	III. THE FIRST BREATH OF AIR

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With the assumption that a mutant mouse is born without lethal morphological, vascular, or hemostatic defects, its first extrauterine challenge will be to breathe. When the pups are being born, they remain cyanotic for a few minutes. This is a normal phase corresponding to the period of time between the arrest of placental oxygenation and the moment where breathing takes over. After a few breaths of air, the newborns acquire their characteristic pink color. However, the first breaths of air are critical, and a large number of life-threatening anomalies that affect different physiological systems, in addition to the lungs, can interfere with normal breathing or lead to poor oxygenation of the blood (Table 1).

In mice, lung development is a complex process that can be divided into four specific stages (14). At the pseudoglandular stage (E9.5–E16.5), branching morphogenesis generates the respiratory tree. The canalicular stage follows (E16.5–E17.5), where the terminal bronchioles expand to form the respiratory ducts and sacs. Lung maturation at the saccular stage (E17.5–P5) is marked by the thinning of the mesenchyme, the differentiation of the specialized type I and II pneumocytes, which are involved in gas exchange and surfactant production, respectively, and apposition of juxtaposed capillaries. Septation of the saccules that give rise to alveoli only starts at postnatal day 5. Although the lungs have no survival value for embryonic development, many genes essential for early lung development are also required for embryonic development, and deletion of these genes sometimes leads to death in utero. However, genes essential for morphogenesis, maturation, or gas-exchange function of the lungs are usually revealed by respiratory distress and neonatal death.

1. Branching morphogenesis

Elegant examples of abnormal branching morphogenesis are provided by disruption of genes involved in epithelial-mesenchymal interactions that govern lung ontogeny. Inactivation of the Wnt7b, Hhip, Fgf9, or Tcf21 genes results in hypoplastic lungs. Interestingly, although disruption of all these genes leads to lung hypoplasia, the developmental basis of the defects differs among mutants as well as their neonatal surviving time. Wnt7b mutant mice die within 10 min after delivery with small and collapsed lungs. Lung hypoplasia is associated with a marked decrease in the proliferation of distal mesenchyme and reduced branching early in development, although specification of the lobulation pattern appears normal. Wnt7b mutants also display a late epithelial maturation defect and pulmonary vascular defects, resulting in vessel rupture and hemorrhage at birth (148). The small size of Hhip mutant lungs is associated with a failure to initiate secondary branching of the primary buds during specification of the pulmonary lobes (Fig. 5 A). However, proximal and distal epithelial differentiation occurs normally in these mutants, which likely explains their ability to survive for a few hours (18). Reduced branching complexity is also responsible for the abnormal small size of Fgf9 mutant lungs (Fig. 5B). Since the morphology and differentiation of lung epithelial cells is preserved in these mutants, the mice are able to inflate their lungs and to breathe for a short period of time before dying (27). Deletion of Tcf21 leads to defective terminal branching, resulting in a lack of distal lung structures that is incompatible with lung inflation after birth (128).

View larger version (80K): [in this window] [in a new window]   FIG. 5. Examples of lung defects associated with respiratory failure at birth. A: abnormal lobulation pattern of the lungs in Hhip knockout mice. The mutant lung contains only 2 lobes (bottom panel) instead of 5 lobes in the control lung (top panel). [Modified from Chuang et al. (18), copyright 2003, with permission from Cold Spring Harbor Laboratory Press.] B: lung hypoplasia in Fgf9 mutant mice. [Modified from Colvin et al. (27), with permission of the Company of Biologists.] C: histology of E17.5 Nfib mutant lungs showing absence of saccular structures (right panel). [Modified from Steele-Perkins et al. (151), with permission from the American Society for Microbiology.] D: differentiation defects of type I pneumocytes revealed by histological examination of instillated Pdpn mutant lungs. Note that the mutant lung (right panel) does not expand as much as the control lung (left panel) after instillation. V, blood vessel; P, pleura. [Modified from Ramirez et al. (129), copyright 2003, with permission from Elsevier.] E: abnormal appearance of secreted surfactant in Sftpb-deficient newborn lungs (right panel) as revealed by electron microscopy analysis. [Modified from Clark al. (19), copyright 1995, National Academy of Sciences, USA.] F: ultrastructure analysis reveals a high glycogen (gl) content in type II pneumocytes of Hspa5 B/B mutant lungs (right panel) compared with control tissue (left panel). Lamellar bodies in mutant type II pneumocytes (arrow) are abnormal. [Modified from Mimura et al. (105), copyright 2007, with permission from Macmillan Publisher Ltd.] G: survival curve of Scnn1a null mutants presenting lung clearance defects. [From Hummler et al. (68), copyright 1996, with permission from Macmillan Publisher Ltd.]

Mutations affecting lung maturation can also challenge normal breathing, as illustrated in the Nfib, Pdpn, and Ndst1 knockout mouse models. Although these mutants make visible efforts to breathe, lung inflation is not sufficient to provide adequate gas exchange in the lungs, and these mutants die within minutes after birth following a few agonal breaths. Lungs from mice deficient in Nfib are stuck at the pseudoglandular stage. They lack saccular structures (Fig. 5C) and, consequently, are deficient in both type I and II pneumocytes (58, 151). Pdpn mutants display abnormal distal saccules associated with a blockade in type I pneumocyte differentiation (Fig. 5D). Interestingly, secretion of surfactant is abundant in mutant lungs, indicating that maturation of type II pneumocytes does occur normally (129). At the opposite, type II pneumocyte maturation is compromised in Ndst1 mutants, resulting in impaired surfactant production. These mice experience severe respiratory difficulties and survive less than 10 h postpartum (41).

Lung maturation is associated with apposition of blood vessels and respiratory epithelium. With the increase in blood flow accompanying birth, the vascular system must be well developed to allow proper gas exchange. Mouse newborns deficient for the gene Nos3, which encodes endothelial nitric oxide synthase, die within the first hour of birth from severe respiratory distress. Striking abnormalities in pulmonary vascular development are observed in these mutants, characterized by a reduction of distal arteriolar branches and marked capillary hypoperfusion. The lung pathology in these mutants resembles that of human infants with alveolar capillary dysplasia (60).

Lung maturation can also be delayed by non-cell-autonomous factors such as hormonal deficiencies. Glucocorticoid deficiency in the Crh mutant or absence of the glucocorticoid receptor (Nr3c1 knockout) results in delayed lung maturation, and mutant mice die a few hours after delivery (23, 113). These examples show how hormonal defects can affect lung development and neonatal survival.

3. Transition to air breathing

Inadequate gas exchange in a morphologically normal lung can also impair neonatal respiratory functions. Successful adaptation to air breathing requires the appropriate production of surfactant from the mature lung, and clearance of the liquid in the lungs after birth. The prototypical example of surfactant deficiency is the deletion of the Sftpb gene, which encodes surfactant protein B. In these mutants, respiratory failure associated with atelectasis results from the disruption of storage, routing, and function of surfactant lipids. In the absence of functional surfactant (Fig. 5E), which decreases surface tension of the respiratory saccules, the lungs cannot inflate, and the animals remain cyanotic and die within minutes (19). Another example is provided by a specific mutation in the Hspa5 gene, which encodes the endoplasmic reticulum chaperone BiP. Mice expressing a mutant BiP protein that lack the retrieval KDEL sequence (Hspa5 B/B) display shallow breathing and die within hours of birth, due to impaired biosynthesis of surfactant. Administration of perfluorocarbon, a substitute of surfactant, into the oropharynx improves lung inflation and neonate activity of Bip mutants (105). The defective biosynthesis of surfactant is associated with high glycogen content in Hspa5 mutant lungs (Fig. 5F), a histological feature typical of type II pneumocytes immaturity (133).

In addition to surfactant deficiency, inadequate lung clearance after birth is also associated with low survival of newborn mice. This process is dependent on Na⁺ absorption (120). Mice with a disruption of Scnn1a, encoding the -subunit of the epithelial sodium channel (ENaC), are able to breathe and inflate their lungs after birth but die during the following 40 h (Fig. 5G). Defective lung clearance in Scnn1a mutant mice results in liquid reaccumulation in the lungs a few hours after birth, which interferes with adequate lung inflation (68).

4. Upper airways

In addition to lung specific defects, obstruction of the upper airways can be a cause of respiratory distress. Nasal obstruction is incompatible with normal breathing, since during attempted respiration, the tongue is pulled to the palate, resulting in obstruction of the oral airway. This leads to respiratory distress and death from asphyxia. A mouse mutant presenting this malformation (choanal atresia) was described for a gene involved in retinoic acid biology. Aldh1a3 null mutants show respiratory distress, are cyanosed, and die within 10 h after birth (40).

Mouse mutants presenting neonatal lethal phenotypes have been of great value in revealing the function of genes essential for lung development, maturation, and function. In addition, these mutants represent valuable models of congenital lung abnormalities and respiratory distress syndrome in humans (22, 163).

B. Cardiovascular Defects

The heart is the first organ to form in the embryo, and the establishment of a normal circulatory function is a prerequisite for embryo survival. In humans, congenital heart defects are the leading cause of perinatal lethality (81). The heart develops from two populations of cardiac mesodermal progenitors. Cells within the first heart field contribute to the left ventricular myocardium, whereas the second heart field contributes to the right ventricular myocardium, endocardium, and outflow tract (10). Histologically, there is no difference between the fetal and the postnatal heart. However, with the initiation of breathing at birth, dramatic changes occur in the circulatory system. The heart must also be strong enough to sustain the increased charge associated with newborn activity. Many targeted mutations in mice produce cardiovascular defects, which are largely responsible for in utero embryonic lethality (29). In this section, we discuss typical cardiac defects that bypass embryonic lethality but are incompatible with the establishment of a normal respiratory circulation. These cardiovascular abnormalities are associated with poor blood oxygenation, resulting in congenital cyanosis, respiratory distress and, inevitably, neonatal death. The interested reader is referred to a recent review for a more detailed discussion of cardiovascular defects leading to preterm lethality (29).

1. Outflow tract and aortic arch

Studies of neonatal lethal mutants have been particularly useful for the understanding of congenital heart diseases in humans, especially in the identification of genes involved in outflow tract formation (57). Aortic arch anomalies were observed in Foxc2 mutant mice, which show gasping motion, cyanosis, and collapsed lungs, resulting in death within 10 min of birth (69). Ablation of the Sema3c gene leads to severe cardiovascular malformations characterized by interruption of the aortic arch and persistent truncus arteriosus (Fig. 6 A). These mutant mice are cyanotic and die shortly after birth (42). Endothelial defects leading to outflow tract abnormalities were also described for a mutant of the Plxnd1 gene (Fig. 6B). Interestingly, the related phenotypes of Sema3c and Plxnd1 null mutants have helped integrating their gene products into a common signaling pathway important for vascular patterning (52). Similarly, characterization of mice conditionally inactivated for Gata6 in neural crest cells revealed an epistatic relationship between Gata6 and Sema3c for outflow tract and aortic arch development (93). These examples illustrate the importance of phenocopies of neonatal lethal mouse mutants in gene function studies.

View larger version (83K): [in this window] [in a new window]   FIG. 6. Cardiac defects compromising neonatal survival in newborn mice. A: aortic arch anomalies in Sema3c mutant mice. Closure of the ductus arteriosus (yellow in inset) after birth disrupts the systemic circulation in mutant hearts. [Modified from Feiner et al. (42), with permission of the Company of Biologists.] B: only one great vessel, the truncus arteriosus (TA), arises from the heart of mice deficient for the Plxnd1 gene. Pulmonary circulation is disrupted in these mutants. Notice the normal closure of the ductus arteriosus in wild-type mice. Ao, aorta; PA, pulmonary artery. [Modified from Gitler et al. (52), copyright 2004, with permission from Elsevier.]

A frequent type of cardiovascular defect challenging neonatal survival involves the failure of cardiovascular remodeling after birth. With the initiation of respiration in the newborn, the transition from fetal (placental) circulation to respiratory circulation (increased blood flow in the lung) is dependent on the closure of a specialized vessel called the ductus arteriosus. The ductus arteriosus connects the pulmonary artery to the aorta and shunts pulmonary circulation to the systemic circulation in the fetus. Failure of the ductus arteriosus to close after birth threatens neonatal health as shown by respiratory complications in human newborns presenting a patent ductus arteriosus (160). Initial closure of the ductus arteriosus is mediated by contraction of its thick muscular wall within 30 min after delivery with functional closure by 3 h (154). Genetic studies in the mouse have confirmed the importance of prostaglandins in the biology of ductus arteriosus closure. Deletion of Ptger4, which encodes a receptor for prostaglandin E₂ (PGE₂), leads to a defect of ductus arteriosus closure. Although initial ductal closure occurs in these mutants, they die between 24 and 48 h after birth from lung edema and congestive heart failure due to a reopening of the ductus arteriosus (119, 141). As expected, deficiency in the synthesis of PGE₂ also leads to a patent ductus arteriosus in mice deficient in prostaglandin dehydrogenase (Hpgd gene), which die 12–24 h after birth (21). Other deletion studies of the cyclooxygenase genes Ptgs1 and Ptgs2 show that patent ductus arteriosus results in labored breathing, cyanosis, and death as soon as 30 min after birth, with no animals surviving for more than 12 h (98). These examples illustrate the importance of cardiac remodeling after birth for survival of newborn mice.

C. Neuromuscular Defects

Breathing is a physiological process governed by the brain that involves a rhythmic activity produced by a neural circuitry that develops before birth and is responsible for immediate breathing of the newborns. The respiratory center resides in the lower brain stem and comprises rhythm generator neurons of the parafacial respiratory group (pFRG) and pattern generator neurons of the ventrolateral medulla (VLM). The respiratory rhythm is transmitted through the spinal motoneurons to the respiratory muscles: the diaphragm and intercostal muscles (132). Defects in any of these structures can lead to respiratory failure.

1. Nervous system

Specific defects in the respiratory center lead to inadequate respiratory activity, and a number of neonatal lethal mutants have been instrumental in the characterization of these structures. Inactivation of Egr2, which encodes the transcription factor Krox-20, is associated with breathing instability and death a few hours after birth. The study of these mutant mice not only showed the importance of this gene for the control of hindbrain segmentation, but also revealed the importance of proper hindbrain segmentation in the development of the respiratory rhythmogenic neuronal network (73, 152). In the Atp1a2 knockout mouse model, persistent depolarization of neurons in the brain stem results in the absence of respiratory activity (Fig. 7 A). These mice are akinesic, present gasping-like respiration, and do not survive more than 10–15 min after birth (70, 112). On histological examination, the lungs of Atp1a2^–/– embryos at E18.5 appear developmentally normal, but the saccules are less expanded (Fig. 7B) (112). Neonatal death due to apnea can also result from abnormal neuronal oscillation patterns. Mice deficient for the Mafb gene suffer from central apnea, show gasping behavior, and die within 2 h after delivery (4). Abnormal activity of inspiratory neurons in the medulla also leads to central respiratory failure (Fig. 7C) and neonatal death in Tlx3 (147) and Pbx3 null mice (131), which represent models for congenital central hypoventilation syndrome. Depression of the respiratory center as a result of excessive glycinergic neurotransmission was described in the Slc6a9 knockout mouse (Fig. 7, D and E), a model of human glycine encephalopathy syndrome. These mice die between 6 and 14 h after birth due to respiratory distress. In addition, the inability to feed in association with bradykinesia was observed in these animals (Fig. 8 A) (54, 157). These mouse mutants have proven valuable models to study the developmental disorders of respiratory control in human (48).

View larger version (73K): [in this window] [in a new window]   FIG. 7. Investigation of abnormal breathing activity in mutant mice. A: example of inspiration-related activity recorded from the C4 ventral root of isolated brain stem preparation of E18.5 fetuses. Notice the absence of spontaneous respiratory rhythm activity in Atp1a2 homozygous mutant mice (bottom). [Modified from Ikeda et al. (70), copyright 2004, by the Society for Neuroscience.] B: histological analysis showing that the saccules of Atp1a2 mutant lungs (right panel) are less dilated than wild-type controls (left panel) consequent to the failure of normal prenatal breathing activity. [Modified from Moseley et al. (112), with permission from the American Society for Biochemistry and Molecular Biology.] C: apnea in newborns can be revealed by sudden cyanosis. Notice the disappearance of cyanosis after mechanical stimulation of the same Tlx3 homozygous mutant mouse (arrow). [Modified from Shirasawa et al. (147), copyright 2000, with permission from Macmillan Publisher Ltd.] D: whole body plethysmography recording as a measure of in vivo respiratory activity in neonates. Traces reveal a severely depressed respiratory frequency in Slc6a9 mutant mice (bottom). E: breathing patterns in Slc6a9 mutant newborns. Note the depression of respiratory frequency (left panel) and prolonged expiratory interval (right panel) in homozygous mutants. [Modified from Gomeza et al. (54), copyright 2003, with permission from Elsevier.]

View larger version (92K): [in this window] [in a new window]   FIG. 8. Phenotypic appearance of mouse newborns presenting neuromuscular defects. A: dropping forelimbs and abnormal body curvature indicative of a neuromuscular defect in Slc6a9 knockout mice (right panel). [Modified from Gomeza et al. (54), copyright 2003 with permission from Elsevier.] B: Kif1b mutant newborns present a similar abnormal appearance (right panel). [Modified from Zhao et al. (174), copyright 2001, with permission from Elsevier.] C: Slc5a7 homozygous mutant newborns exhibit dropping forelimbs and are markedly cyanosed, indicative of respiratory distress. [Modified from Ferguson et al. (44), copyright 2004 National Academy of Sciences, USA.] D: abnormal curvature of Chat mutant fetuses associated with NMJ defects is visually observable as soon as E16.5. [Modified from Misgeld et al. (107), copyright 2003 by the Society for Neuroscience.] It is interesting to note that the disruption of genes involved at various levels of motor function can result in similar phenotypic appearance in newborn mice.

2. Neuromuscular junction

Proper muscle contraction to allow breathing is dependent on the development of a functional neuromuscular junction (NMJ). Genetic analysis in mice has provided key insights into the molecular control of neuromuscular synaptogenesis (89). A defect at the NMJ is sufficient to cause neonatal lethality, as exemplified by the Musk and Agrn gene mutants. In the absence of any of these genes, the NMJ does not form, leading to akinesia. The lungs remain collapsed due to the inability to breathe, and mutants die immediately after birth (33, 49). The phenocopy of these two mouse models has helped understand the receptor-ligand relationship of the MuSK and Agrin proteins and their role in NMJ formation. Normal synaptic transmission at the NMJ is also important for respiration, as revealed by the inactivation of the Slc5a7 gene that encodes a choline transporter. Insufficient acetylcholine release at the NMJ of these mice results in a limited capacity of movement, dropping forelimbs (Fig. 8C), and death within 1 h of birth from respiratory distress (44). The absence of clustering of acetylcholine receptors at the NMJ resulting from disruption of the Rapsn gene leads to a similar phenotype, and these mice die a few hours after birth from weak breathing (50).

3. Respiratory muscles

The inability to support the dramatic increase in muscular activity required for respiration immediately after birth leads to respiratory distress and neonatal death. Therefore, mutations affecting muscle development as well as muscle function can challenge neonatal survival. The classical example is the targeted deletion of Myog, which encodes the muscle regulatory factor myogenin. These mutant mice are unable to generate a force sufficient for respiration due to a severe reduction of respiratory muscles such as the diaphragm (Fig. 9A). They are immobile and cyanosed after birth and die immediately (62, 115). Atp2a1 encodes a sarcoplasmic reticulum Ca²⁺-ATPase isoform (SERCA1) that is essential for the function of fast-twitch type II muscular fibers. Mice deficient for this gene die of respiratory distress due to abnormal contraction of the diaphragm. They show gasping respirations, have limited chest wall and limb movements, and die within 2 h after birth (122).

View larger version (95K): [in this window] [in a new window]   FIG. 9. Typical examples of muscular and skeletal defects associated with respiratory failure at birth. A: histological examination of muscle diaphragm in E18.5 embryos. Notice the thin diaphragm that lacks myofibers in Myog mutant mice (right panel). [Modified from Knapp et al. (82), with permission from the Company of Biologists.] B: skeletal preparations showing a smaller rib cage (dorsal view) in Tbx18-deficient newborn mice (right panel). Note the ossification band in the proximal region (arrows) and fusion of distal ribs (arrowhead) in Tbx18 mutant. [Modified from Bussen et al. (12), copyright 2004, with permission from Cold Spring Harbor Laboratory Press.]

D. Skeletal Defects

The skeleton is made of two distinct tissues: cartilage and bone. Unlike other organs, it spreads throughout the entire body and is composed of more than 200 elements of various shapes (80). It is not surprising that abnormal skeletal development in mutant mice can result in a wide spectrum of phenotypic outcomes ranging from slight anomalies to catastrophic damage of the fetus. Since a large number of these mutations are associated with death at birth, analysis of neonatal lethal phenotypes has therefore been useful to identify genes important in various aspects of skeleton biology such as patterning and chondrogenesis (26, 111). Mice deficient for Runx2, a candidate gene for cleidocranial dysplasia syndrome in humans, illustrate well the importance of the skeletal system for neonatal survival. The skeleton of these mutants lacks ossification (bones), with the consequence that newborns show gasping respiration and die within 20 min after birth (84, 121). Skeletal defects can threaten neonatal survival in various ways such as by failing to protect the newborn during parturition (leading to lethal damages) or by interfering with normal breathing.

1. Rib cage

The rib cage participates in respiration principally by providing structural support to respiratory muscles. One would predict that defects in the rib cage would lead to respiratory distress and neonatal death. Indeed, absence of the rib cage, as initially described for Myf5 knockout mice, is incompatible with neonatal survival (6). Targeted deletion of various genes involved in the induction of skeletal structures, skeletogenesis, or chondrogenesis is associated with abnormal rib cage formation and neonatal death. Uncx4.1 is a homeobox gene found to be important for specification of skeletal elements derived from the lateral sclerotome, such as the proximal ribs. Null mutants have a distorted and smaller thoracic cavity, which is associated with poor inflation of the lungs after birth. These mice present kyphosis (severe lordosis) and die shortly after birth (92, 100). This phenotype is reminiscent of mutants with a Tbx18 deletion that present an abnormally small rib cage (Fig. 9B) and die rapidly after a few irregular breaths (12). A dismorphic rib cage is associated with respiratory failure and neonatal death in mice deficient for Ctgf, a gene essential for matrix remodeling during chondrogenesis (72).

Similar to lung development, hormonal deficiency can also affect skeletal development, as shown by inactivation of the Pthlh gene, which encodes the parathyroid hormone-related peptide. Pthlh homozygous mutant mice die immediately after birth from respiratory distress due to abnormal ribs and sternum (79).

2. Vertebrae

Although a damaged rib cage can intuitively lead to respiratory distress at birth, a number of mouse mutants present vertebral defects rather than rib malformations. Typical examples are the Bapx1 and Col2a1 gene mutations that result in lethal skeletal dysplasia. These mutants exhibit abnormal vertebrae, lack intervertebral discs, and die immediately after birth from respiratory failure, even though they have normal ribs (1, 94, 96). It cannot be excluded that such skeletal defects can interfere with spine, nerves, or even normal brain functions associated with breathing. However, unless neural defects are obvious, such as dorsal root ganglia fusion in the Uncx4.1 mutant (92), the integrity of the spine and nerves is usually not investigated in skeletal mutants. As a consequence, neuronal malfunction could account for neonatal lethality in some cases of skeletal phenotypes. Nevertheless, neonatal lethal phenotypes have been useful to reveal the function of genes essential for skeletal development that represent potential candidates for human skeletal genetic disorders (97).

	IV. ABNORMAL FEEDING

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Little or no milk in the stomach a few hours after birth is indicative of abnormal feeding (Fig. 10 A). Complete inability to feed will obviously result in neonatal death due to the absence of nourishment, but also because the liquid derived from the milk is essential for homeostatic processes in newborns (123). Its absence leads to dehydration, and intraperitoneal injection of saline usually rescues survival for several hours. Analysis of a number of neonatal lethal mutant mice has revealed that nonfeeding or normal fasting newborns die within a window of 12–24 h after birth (34, 110, 143). Therefore, if death occurs before this period, it is unlikely that problems related to abnormal feeding are the primary cause of death. Feeding problems can result from abnormal nursing or rejection by the mother. However, the major causes of the inability to feed in mutant mice are associated with neuromuscular or craniofacial skeletal defects (Table 2).

View larger version (53K): [in this window] [in a new window]   FIG. 10. Suckling defects in mutant mice. A: absence of milk in the stomach (white patch in wild-type mice) of a Cntfr mutant newborn (top panel). Cntfr-deficient mice die between 12 and 24 h after birth in the absence of nourishment (bottom panel). [Modified from DeChiara et al. (34), copyright 1995, with permission from Elsevier.] B: lack of movement coordination is evident in Pou4f1 mutant newborns that cannot use their forearms to right themselves (arrow). Notice the white spot of milk in control mice (arrowhead). [Modified from Xiang et al. (171), copyright 1996 National Academy of Sciences, USA.] C: cleft of the secondary palate in Lhx8 mutants (arrowheads in right panel). [Modified from Zhao et al. (176), copyright 1999 National Academy of Sciences, USA.]

Suckling is a complex process that involves many structures of the brain, nerves, as well as the muscles required to extract milk (5). Olfactants on the nipple are the main sensory cue used in orientation, whereas tactile sensation is required to initiate the suckling reflex. The suckling response includes nipple attachment, suckling with rhythmic movement of the jaw and tongue, and the stretch response (167). These processes are regulated by complex interactions between sensory and motor neuronal pathways, which are linked to the central nervous system through the brain stem trigeminal complex. Motor neurons involved in feeding are located in the facial motor nucleus (face), trigeminal motor nucleus (jaws), and hypoglossal motor nucleus (tongue) (78). Therefore, defects in any of these structures are likely to impair suckling and challenge neonatal survival.

1. Olfaction

In newborn rodents, olfaction is essential for normal suckling behavior (134). The importance of olfactory integrity for suckling is well illustrated by the knockout of Edg2 gene, which encodes a lysophosphatidic acid receptor. These mutant mice present developmental abnormalities in the olfactory bulb and cerebral cortex, resulting in 50% neonatal lethality due to impaired suckling behavior. Some of the mutant pups can survive for a few days after birth depending on the milk content in the stomach, thereby establishing a link between olfaction, milk content, and survival (28). Similarly, targeted inactivation of Ebf2, a member of the repeated helix-loop-helix transcription factor gene family, leads to defects in the projection of olfactory neurons to the dorsal olfactory bulb and is associated with neonatal lethality (165). Severe loss of olfactory and autonomic neurons was described in mice deficient for the Ascl1 gene, which do not survive more than 24 h after birth (59). This survival time is shorter than that of anosmic mice. Mutants for the genes Cnga2 and Gnal, which are involved in olfactant signal transduction, fail to exhibit any odorant-evoked excitatory response and die within 2 days from lack of feeding (3, 9). It is interesting to note that in some cases of Gnal mutants, litter depletion increases survival of the mutant pups, by opposition to Fyn knockout pups, which benefit from suckling littermates to stimulate the mother's nipples to be able to feed (172). Although suckling is an olfactory-driven behavior, the timing of death as well as the partial survival of anosmic mice suggests that olfaction is more important in suckling maintenance than in the initial suckling reflex.

2. Sensory-motor

Suckling, like breathing, is regulated by the brain stem. It is therefore not surprising that defects in hindbrain development which affects respiratory rhythm, such as in the Egf2 mutant (Table 1), also affect suckling activity. It is likely that such anomalies of the brain stem will result in respiratory failure. However, specific defects in this region that affect suckling can also challenge neonatal survival. A classical example is provided by the ablation of the Cntfr gene, which encodes a receptor for ciliary neurotrophic factor. Homozygous mutants die within a window of 12–24 h (Fig. 10A) and cannot open their jaws, resulting in the inability to suckle. Lethality in these mutants is associated with severe defects in motor neuron populations, including those located in the brain stem nuclei associated with suckling (34).

The suckling behavior can be affected by sensory neuron deficits. These defects are revealed by the inability of mutant pups to reach the nipple, to open their jaws to suckle, or both. Neonatal lethal mouse mutants have helped to identify genes involved in sensory neuron development, such as Grin2b, which encodes the NMDA receptor -subunit. Homozygous Grin2b mutants show severe sensory defects and die within 24 h, even if their mother nurses them normally (90). The primary sensory neurons do not interact with the brain stem trigeminal complex in these mice, explaining why sensory input does not induce suckling movement in mutant pups. A milder phenotype was described for Bdnf (brain-derived neurotrophic factor) knockout mice, which can survive up to 2 days with sensory neuron dysfunction (76).

The importance of the motor system is well illustrated by the knockout of Pip5k1c, which encodes a phosphatidylinositol-4-phosphate 5-kinase essential for synaptic vesicle trafficking. The inability to feed in these mice is due to hypoactivity and results in death within 24 h (37). Defects at the NMJ are also associated with the inability to feed, although the majority of the mutations affecting NMJ function lead to respiratory failure (see above). For example, inactivation of the Chrng gene, which encodes a fetal-type acetylcholine receptor, results in impaired breathing that compromises neonatal survival. However, mutants able to breathe, even irregularly, cannot feed and die within 2 days. These mice cannot move their hindlimbs and present the characteristic lunar shape of mouse mutants with neuromuscular defects (155). In the Pou4f1 mutant, motor defects are more severe, and both the inability to open their jaws as well as characteristic uncoordinated limb and trunk movements are responsible for abnormal feeding in these mice (Fig. 10B) (103, 171). An additional example is provided by mice with a targeted deletion of Nr2f1, which display abnormal swallowing reflex and cannot survive more than 36 h after birth. Defects in morphogenesis of the glossopharyngeal nerve resulting in inappropriate innervation of the pharynx and root of the tongue is likely responsible for the feeding problem of these mutants (127). These examples illustrate the importance of peripheral sensory and motor neuron integrity for suckling and neonatal survival.

3. Hypothalamus

The appetite of newborn pups can also influence the amount of milk intake. Ablation of the Hap1 gene leads to early postnatal death, and a correlation was made between milk intake and survival. Tactile sensation and motor control are normal in these mice (16, 39). More recent work has revealed that reduced expression of Huntingtin-associated protein 1 decreases the levels of GABA_A receptors in hypothalamic neurons and causes a decrease in food intake, explaining the abnormal feeding behavior of Hap1 mutant mice (145).

4. Muscles

Neuronal defects are the prevailing cause of abnormal feeding behavior in mutant mice. Although muscular deficiencies can be associated with defective innervation, suckling impairment can also result from cell-autonomous muscular defects. For example, mouse mutants for Jph1, which encodes the transmembrane protein junctophilin, display an abnormal triad junction formation in the skeletal muscles of the face. This results in the inability to suckle due to impaired contractile activity of the jaws (71). However, such examples are rare, since most mutant mice presenting muscle phenotypes are likely to die of respiratory failure as described above.

B. Craniofacial Defects

Abnormal suckling can be caused by skeletal defects leading to facial malformations, the most common being a cleft of the secondary palate. Absence of the secondary palate in the mouth leads to failure to suckle because of the absence of negative pressure in the oral cavity during the suckling process. Analysis of mutant mice has demonstrated that a cleft palate can result from an intrinsic defect in palatal shelves fusion or as a consequence of the abnormal development of craniofacial structures (45, 65).

The Lhx8 knockout mouse is a good example of the importance of normal palatogenesis for neonatal survival. Disruption of Lhx8, which encodes a homeobox transcription factor expressed in palatal mesenchymal structures, results in a phenotype of cleft palate (Fig. 10C); the penetrance of the defect correlates well with neonatal death (176). Other examples of intrinsic defects that lead to abnormal palate formation are provided by the disruption of the Osr2 and Tgfb3 genes. Failure of the palatal shelves to fuse in the Tgfb3 mutant results in a cleft palate, and death occurs within the normal time range for nonfeeding mice (77). Osr2-deficient mice also die within 24 h, although in this case reduction of palatal mesenchymal cell proliferation is responsible for the cleft palate (91). Isolated cleft palate is rare in mouse mutants, and a large number of cleft palate phenotypes result either from abnormal tongue development, such as in Foxf2 mutant (166), or develop secondary to craniofacial defects. Among the latter group are mutations in the Maguk family genes Dlgh1 and Cask (15, 169) and the homeobox genes Hoxa2, Msx1, and Dlx2 (168). These mutants show inadequate development of neural crest derivatives.

It should be noted that neonatal death of certain mutants that present a cleft palate is not exclusively due to abnormal feeding but may be secondary to respiratory distress. For example, Foxf2 null mutants die within 18 h after birth because of an abnormally air-distended gastrointestinal tract (166), a phenotype also observed in Pax9 mutant mice (125). Air distention of the abdomen likely leads to interference with normal breathing or with blood supply to the visceral organs and ultimately results in rupture of the gut. A different example is the fusion of the tongue with the palatal shelves in Jag2 mutants that is associated with respiratory distress and early death of the newborns (75). Therefore, the inability to suckle due to a cleft palate is not always the principal cause of death. Nevertheless, neonatal lethal mouse mutants presenting palatal defects represent useful models of cleft palate, which is a frequent malformation in human newborns (67).

C. Parental Behavior

The newborns are totally dependent on their mother, and the consequence of abnormal nursing behavior is inevitably neonatal death. Although rodent pups have some form of resistance to hypothermia, their survival outside a warm nest is greatly compromised. Analysis of a number of mutant mice suggests that the time of death of pups that are kicked-out of the nest by their mother is probably shorter than that of nested nonsuckling newborns, although determining the precise time of death for fasting newborns outside a controlled environment is difficult because of ethical issues. As in many mammals, normal nursing by the mother is dependent on the recognition of babies through vocalization. Absence or diminished vocalization due to inability to open the jaws could result in rejection by the mother.

	V. HOMEOSTASIS DEFAULTS

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During pregnancy, homeostasis of the fetus is critically dependent on the placenta, which is responsible for nutrient supply, metabolic exchange, and waste disposal. After birth, the newborn must adapt quickly to new metabolic needs and regulate its own homeostasis. Many mutations in mice that interfere with the state of equilibrium of various cellular functions and the chemical composition of fluids and tissues are likely to remain silent during embryonic development but can seriously challenge neonatal survival. Neonatal lethal phenotypes in mice have helped reveal the function of genes involved in energy homeostasis, fluid-electrolyte balance, and metabolism, principally through symptoms such as hypoglycemia, dehydration (transepidermal water loss), and renal failure (Table 3).

Glucose is an important energy source for neonates, which are in sudden demand of energy for their metabolic requirements. Soon after delivery, there is a period of starvation that follows the cessation of nutrient supply provided by the maternal circulation. Gluconeogenesis is not fully active in the immediate postnatal period (51). During the period that precedes suckling, glucose must be produced to combat hypoglycemia mainly by glycogenolysis and mobilization of glycogen stores from the liver. Indeed, late in gestation, glycogen synthase expression is induced in the liver to promote the storage of glycogen that will allow survival of the newborn during postnatal starvation (32). Hormonal control of this adaptation involves increased production of glucocorticoids, decreased insulin levels, and secretion of glucagon (36). The newborns must also adapt to self-nourishing and regulate energy utilization between feedings. Therefore, mutations that disrupt glucose homeostasis as well as normal development of the liver or pancreas are life threatening for the newborn because they affect energy balance.

Following birth, transient hypoglycemia occurs until glycogenolysis and gluconeogenesis are activated in the liver (32). A critical source of energy immediately after birth is obtained through autophagy (85). The study of Atg5 knockout mice, which die neonatally, was instrumental to understand the importance of autophagy for neonatal energy needs. With the fall of glucose levels after birth, autophagy is induced in response to starvation and reaches a maximum at 3–6 h postpartum. Autophagosome fusion is blocked in Atg5 homozygous mutants, and these mice die within 12 h of birth, with their energy levels severely depressed (88). A similar phenotype and survival time are observed upon disruption of Atg7 gene (Fig. 11 A), confirming its role in early autophagy (83). Analysis of these mutants has highlighted the essential role of autophagy in providing energy to sustain life during the critical period of hypoglycemia that accompanies the transition from transplacental nutrition to milk.

View larger version (59K): [in this window] [in a new window]   FIG. 11. Energy homeostasis defects are associated with poor neonatal survival. A: appearance (left panel) and survival curve (right panel) of autophagy-deficient Atg7 mutant newborns in the absence of feeding. [Modified from Komatsu et al. (83), with permission from the Rockefeller University Press.] B: periodic acid-Schiff (PAS) staining reveals decreased liver glycogen content at term in Eif2s1 (A/A) mutant mice. C: blood glucose levels under nonfeeding, fasting conditions in Eif2s1 mutant mice littermates. Notice that A/A mutants cannot increase their blood glucose after birth (left panel) and remain severely hypoglycemic after delivery (right panel). [Modified from Scheuner et al. (139), copyright 2001, with permission from Elsevier.]

Hypoglycemia in the newborn is sometimes associated with cyanotic episodes, respiratory distress, and abnormal feeding. For instance, mice with a targeted deletion of the Hmgb1 gene show a moderate glucose homeostasis defect leading to early hypoglycemia. Soon after birth, these mice become lethargic and suckle very little, resulting in death within 24 h despite normal nursing by the mother (13). Hypoglycemia, as a result of increased insulin sensitivity, is also associated with cyanosis and lethargy in mice with a mutation disrupting the Ship2/Phox2a loci (20). Abnormal suckling behavior must therefore be carefully analyzed in mutant mice neonates as it could be the consequence of an underlying energy homeostasis defect.

B. Transepidermal Water Loss

The epidermis provides a water-impermeable barrier that prevents excessive loss of body fluids, a function that is critical for survival after birth. Impermeability is normally fully developed by E17 in the mouse (61). The barrier function of the skin is conferred by a combination of the cornified envelope and intercorneocyte lipids within the outermost layer of the epidermis that work as bricks and mortar. Development of this specialized structure, called the stratum corneum, involves the formation of junctions between keratinocytes, accumulation of specialized keratin, formation of the cornified envelope, and extrusion of specialized intercorneocyte lipids (118). Gene mutations affecting skin development at any of these steps can result in transepidermal water loss and seriously affect neonatal survival.

The importance of skin barrier function for neonatal survival is well illustrated by the Spink5 gene deletion, which is associated with Netherton syndrome in humans (35, 64). Deficiency of this gene, which encodes the serine protease inhibitor Lekt1, leads to degradation of desmoglein 1 as a result of epidermal protease hyperactivity. While the skin appears normal at birth in Spink5 mutant mice, spontaneous detachment of the stratum corneum is observed within 30–60 min, resulting in large skin erosion (Fig. 12 A). The important loss of water explains the short 2-h survival time of these mutants. A severe skin phenotype is also observed upon inactivation of Tgm1, which represents a model for human lamellar ichthyosis (101). Deletion of this gene, which encodes transglutaminase 1, an enzyme that cross-links the cornified envelope, results in severe skin defects associated with an abnormal shiny skin (Fig. 12B) and dehydration of the body extremities. Tgm1 null mutants are less active than control littermates and die within 5 h postpartum.

View larger version (89K): [in this window] [in a new window]   FIG. 12. Characteristic appearance of mouse newborns with defects in skin barrier function. A: shedding of the outer layer of the skin in Spink5-deficient newborns. [Modified from Hewett et al. (64), copyright 2005, with permission from Macmillan Publisher Ltd.] B: erythematous and taut skin appearance of Tgm1 mutant newborns. [From Matsuki et al. (101), copyright 1998 National Academy of Sciences, USA.] C: wrinkled skin appearance in Cldn1 mutant newborns. D: neonatal loss of weight associated with transepidermal water loss is a diagnostic feature of skin barrier defects in Cldn1 mutants. [Modified from Furuse et al. (46), with permission from the Rockefeller University Press.]

C. Renal Failure

Neonatal lethality in mouse mutants has also allowed the identification of several genes important for the development and function of the kidney, an organ that plays a crucial role in the regulation of fluid homeostasis. In fact, a large number of genes involved in genitourinary development were revealed by molecular analysis of mutant mice with neonatal lethal phenotypes. Kidney development involves reciprocal interactions between the nephric duct epithelium and the metanephric mesenchyme. Signals from the mesenteric mesenchyme induce ureteric bud evagination from the nephric duct and its subsequent branching. In turn, newly formed ureter tips induce epithelial transition of the mesenchyme and differentiation into nephrons. The branches of the ureter give rise to the collecting ducts (66). The importance of the genitourinary system for neonatal survival was clearly demonstrated in mice deficient for the homeobox gene Emx2, which lack all genitourinary structures and die soon after birth (108, 124).

Defects in development of the kidney proper, ranging from kidney agenesis to subtle defects with associated kidney failure, can seriously compromise neonatal survival. Newborn mice with kidney anomalies have been shown to die during the first 2 days of life, with the timing of death dependent on the severity of the defect. Inactivation of Hs2st1, Wnt4, or Ret genes, which are essential for early development of the kidney, leads to kidney agenesis. Mice bearing these mutations do not survive for more than 24 h after birth (11, 140, 150). Similarly, mice deficient for Itga8, an integrin family member, die from kidney agenesis or dysgenesis; survival time in these animals is dependent on the number and integrity of the remaining kidneys (114). Itga3 mutant mice feed normally but progressively become weaker and dehydrated due to severe glomeruli defects, resulting in death within 24 h of birth (86). Deletion of Pou3f3 results in small kidneys and death within 36 h. These mice exhibit severe defects in the development and function of the nephron, which explains the decreased volume of urine observed as soon as 2 h after birth (117). Anomalies in the development of both the nephron and collecting ducts explain the abnormal appearance of kidneys in mice deficient for Foxd1, which also die within 24 h (63). These examples illustrate how neonatal lethal phenotypes can reveal genitourinary tract defects, especially those affecting kidney ontology.

In addition to defects in kidney morphogenesis, deficiencies in kidney function inhibiting the production and concentration of urine are hazardous to mice (43). Indeed, impaired filtration barrier or electrolyte transport can lead to neonatal death. For example, mice with a disruption of the Nphs1 gene, which encodes nephrin, present no gross morphological defect of the kidney and can feed normally. However, these mice develop massive proteinuria (Fig. 13 A) and edema and die within 24 h after birth. Ultrastructure analysis revealed the absence of podocyte slit diaphragm in Nphs1 mutant mice. These mice provide a model for congenital nephrotic syndrome in humans (126, 130). Anuria is also diagnostic of kidney malfunction. Mice with a disruption of the Podx1 gene are anuric and die of acute renal failure within 24 h of birth. The podocytes of these mice fail to form foot processes and slit diaphragms but instead form impermeable junctional complexes (38). Altered electrolyte homeostasis was described in mice deficient for Scnn1b (Fig. 13B), which encodes the β-subunit of the amiloride-sensitive ENaC. These mutants present hyperkalemia and sodium wasting, and consistently die between 10 and 48 h after birth (Fig. 13C) (102). This phenotype is similar to that of humans with pseudohypoaldosteronism type 1 and provides a model for the pathogenesis of this disorder.

View larger version (32K): [in this window] [in a new window]   FIG. 13. Diagnosis of kidney defects challenging neonatal survival. A: SDS gel electrophoresis analysis of urine samples showing proteinuria in Nphs1 mutant newborns. [Modified from Putaala et al. (126), with permission from Oxford University Press.] B: electrolyte imbalance diagnostic of kidney malfunction in Scnn1b-deficient mice. C: surviving curves of newborn progeny from Scnn1b heterozygous mating. [Modified from McDonald et al. (102), copyright 1999 National Academy of Sciences, USA.]

	VI. CONCLUSIONS

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Only a few organs have a high survival value in utero. In contrast, defects in almost every physiological system appear to seriously challenge survival during the first 24 h of postpartum life in mice. Indeed, the liver, the musculoskeletal system, the lungs, the urogenital system, the skin, and principally the nervous system are highly sensitive to developmental perturbations that can affect neonatal survival. In addition, even minor defects in organs or cellular processes that are essential for embryonic survival such as heart formation, erythropoiesis, vascular and placental development (30), can also impact on neonatal survival. Therefore, a large array of developmental defaults in a number of physiological systems must be considered in the analysis of mouse mutants with no overt phenotype that die neonatally.

The complexity of the analysis of mouse mutants with neonatal lethal phenotypes can be explained by two reasons: 1) a single gene mutation can affect more than one physiological system essential for surviving birth, and 2) abnormalities within a single physiological system can affect more than one physiological process in newborns, including respiration, suckling, or homeostasis, which can be responsible for the lethality observed during the first 24 h. Nevertheless, with a precise determination of the time of neonatal death and an understanding of the various systems important for survival of the newborn, it should become easier to analyze neonatal lethal mouse mutants. In addition to these physiological considerations, special experimental concerns related to this age group must be considered. Fortunately, the repertoire of techniques available for phenotyping mouse neonates is growing and includes sophisticated imaging methods (87). Furthermore, high-throughput phenotyping strategies in mice are being developed (8). The next challenge in neonatal phenotyping analysis will be to adapt these high throughput strategies to this age group.

Analysis of neonatal lethal phenotypes has proven highly informative in dissecting out and analyzing gene functions. First, it allows the identification of genes essential for embryonic processes for which loss of function is only revealed at birth. Second, it obviously identifies genes involved in the development and function of organs or physiological processes essential for extrauterine life. Third, it contributes to the identification of signaling pathways through the analysis of mutant phenocopies. In addition to their contribution to understanding gene function in development, mutants presenting neonatal lethal phenotypes provide extremely valuable models of neonatal syndromes in humans. They are the models of choice for the study of human diseases first occurring during extrauterine life.

	GRANTS

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B. Turgeon is the recipient of a studentship from the Fonds de la Recherche en Santé du Québec. S. Meloche holds the Canada Research Chair in Cellular Signaling. The work performed in the author's laboratory is supported by grants from the Canadian Institutes for Health Research, the National Cancer Institute of Canada, and the Cancer Research Society.

	ACKNOWLEDGMENTS

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We apologize to authors whose original work was not cited directly. All the references for a given gene mutation are easily accessible at the MGI database (www.informatics.jax.org) through the use of the official gene symbols used here.

We are grateful to Drs. Jean Charron, Jacques Drouin, Marc Saba-El-Leil, and Jean Vacher for critical reading of the manuscript. We thank Carol Mathieu for text revision. We also thank all the authors who kindly agreed to provide original manuscript figures.

Address for reprint requests and other correspondence: S. Meloche, Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, PO Box 6128, Station Centre-Ville, Montreal, Quebec H3C 3J7, Canada (e-mail: sylvain.meloche{at}umontreal.ca).

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