Laminin: loss-of-function studies

Laminin nomenclature

Laminin is a large family of heterotrimeric proteins composed of α, β, and γ chains. In mouse and human, 5 α, 4 β, and 3 γ variants have been identified [2]. Different combination of these variants generates a large variety of laminin molecules. Two systems have been developed to name different laminin molecules. The first (old) one involves assigning Arabic numerals to laminins based on the order of their discovery, such as Laminin-1, Laminin-2, Laminin-3, et al. [4]. Although this nomenclature is simple to use, it fails to provide information regarding the trimeric composition of the laminins. Thus, an alternative (new) nomenclature system that indicates each individual polypeptide chain was developed. Specifically, laminins are named by Greek letters representing polypeptide chains followed by numbers indicating the variants [1]. For example: laminin-1 is written as laminin-α1β1γ1 or simply abbreviated as laminin-111. Sometimes, one Greek letter and one number are used to define all laminin isoforms that contain this subunit. For example, “α3 laminins” means all α3-containing laminins.

Some genes encoding laminins may produce more than one peptide due to alternative splicing [5]. For these chains, letters A and B are added after the numbers to indicate the low-molecular-weight and high-molecular-weight peptides, respectively [1]. For example, the gene encoding α3 chain can produce a short laminin α3 variant named α3A and a long laminin α3 variant named α3B [5]. It should be noted that the short α chain variants can be named without mentioning the A letter. Thus, laminin-332 implies laminin-3A32 but not laminin-3B32. A side-by-side comparison of the old and new nomenclature systems is summarized in Table 2.

Laminin structure

Laminin consists of three polypeptide chains (α, β, and γ). Each laminin chain is composed of various globular domains, rod-like tandems or repeats called Epidermal Growth Factor-like “LE” domains, and a coiled-coil domain [1]. The globular domains include laminin N-terminal globular domain (LN), laminin four globular domain (LF), and L4. For example, laminin α1 chain contains a globular LN domain at the N-terminus followed by alternating globular “L4” and LE domains, a coiled coil domain, and five homologous globular “LG” domains at the C-terminus. Compared to the α chain, β and γ chains have similar domains in the N-terminus, but do not have the LG domains in the C-terminus. These chains join together via their coiled-coil domains forming a molecule with one long arm and up to three short arms [1]. The structure of laminin-111 is shown in Fig. 1. The structure of each individual laminin chain is summarized in Table 1. The nomenclature system for these laminin polypeptide chains is well reviewed in other articles [1, 6, 7] and thus not discussed here.

Laminin biochemistry

As a major component of the basement membrane, laminin not only interacts with other extracellular matrix proteins (e.g., nidogen) contributing to basement membrane assembly, but also binds to and activates cell-surface proteins (e.g., receptors) transducing molecular signals. These activities have been mapped to specific domains/regions. In general, sites that mediate laminin-extracellular matrix protein interaction are located in the short arms of all three chains [8]. Laminin-receptor interaction, however, is confined to the N-terminal and C-terminal domains of the α chain [8]. Laminins are usually glycosylated, which increases their molecular masses and stabilizes them against degradation [9]. It should be noted that distinct laminins are not glycosylated to the same extent due to different glycosylation sites [10].

It has been shown that laminin trimers are assembled before being secreted to the extracellular matrix [11, 12]. The assembly starts by combining β and γ chains together, followed by the association of α chain to the β/γ dimer, which is the rate-limiting step [12, 13]. Once secreted, laminins together with other extracellular matrix proteins, including collagen IV and nidogen, form a dense protein network called basement membrane. Although the precise mechanism underlying basement membrane assembly is not fully understood, laminin–nidogen interaction is believed to play an important role in this process [14–17]. The nidogen-binding site on laminin is mapped to a single epidermal growth factor-like domain of the laminin γ1 chain, named γ1III4 [18–23]. It is proposed that nidogen interconnects the two networks formed by laminins and type IV collagen [24], forming a unique 3D structure with high stability. Consistent with this hypothesis, inhibiting laminin γ1-nidogen interaction with antibodies or a recombinant laminin γ1 fragment compromised basement membrane formation in many tissues and organ cultures [25–28]. To further investigate the role of laminin–nidogen interaction in basement membrane assembly, a mouse line (lamc1III4^−/−) with genetic deletion of the nidogen-binding region γ1III4 in laminin γ1 was generated [29]. Interestingly, although the basement membrane in Wolffian duct is compromised [29], skin basement membrane is not affected [30]. These results suggest that laminin–nidogen interaction may play different roles in basement membrane formation/assembly depending on the tissues.

The basement membrane exerts many important functions, including sequestering growth factors and cytokines, establishing concentration gradients and regulating their spatial and temporal distribution [31]. For example, fibroblast growth factors, Wnt factors, and hedgehog are able to strongly bind to the basement membrane. In addition, the basement membrane can also serve as a reservoir for many bioactive fragments usually derived from proteolysis [31]. Furthermore, the basement membrane also participates in ligand maturation/activation. For instance, TGF-β is secreted in its latent inactive form, which is activated by matrix metalloproteinases at the basement membrane [32].

To accurately and timely regulate a variety of critical biological processes, including angiogenesis, the basement membrane undergoes active remodeling via proteolysis [32]. Some laminin chains could be truncated into smaller parts by the action of proteolytic enzymes [7, 11, 33]. The fragments generated by each single enzymatic action are usually named after the acting enzyme. For example: pepsin generates P fragments, trypsin generates T fragments, and elastase generates E fragments [1]. Such enzymatic truncation directly affects the physiological and pathophysiological functions of laminins, and thus the basement membrane [11]. The readers are referred to other excellent reviews for other biochemical properties of the laminin family [8, 34–36].

Laminin function and loss-of-function studies

Laminin exerts many different functions, including embryogenesis, vascular maturation, and neuromuscular development [8, 34, 35, 37–41]. These functions are highly dependent on laminin isoforms, their cellular sources, and developmental stages. Here I discuss and compare loss-of-function studies on each individual laminin chain.

Laminin α1

To investigate the physiological function of laminin α1 chain, a few lama1 loss-of-function mouse lines have been generated. The first lama1 mutant was generated by Miner and colleagues using gene trap technique [42, 43]. Specifically, a gene trap vector was inserted in the third intron of lama1 to produce a fusion protein containing the first 120 amino acids of lama1 and the vector-coded sequences (trans-membrane domain and βgeo). The expression pattern of lama1 (demonstrated by X-gal) in these lama1 trap mice is consistent with previous reports [44–47]. These lama1 trap embryos usually die by embryonic day (E)7 and fail to form Reichert’s membrane [42]. Interestingly, overexpression of lama5, the other laminin α chain (in addition to lama1) expressed in early post-implantation embryos, enables the lama1 trap embryos to initiate gastrulation, but fails to rescue Reichert’s membrane [42]. These studies clearly demonstrate a pivotal role of lama1 in the integrity of basement membranes and early embryonic development.

The second lama1 mutant line was generated by targeted deletion of the proximal promoter region and exon 1 of this gene [48]. Like the lama1 trap mice, these lama1 null mutants die at approximately E6.5, lack Reichert’s membrane, and show defects in parietal and visceral endoderm differentiation [48], again suggesting an important role of lama1 in basement membrane formation and early embryonic development.

Another lama1 mutant line is the LG4–5^−/− mice, which lack the lama1 globular domains 4–5 [49]. Similar to the lama1 mutants described above, these LG4–5^−/− mice die at E6.5 [49]. Although Reichert’s membrane and embryonic basement membranes are grossly normal, defects in epiblast polarization and cavity formation are detected in these mutants [49]. Further studies demonstrate that the truncated lama1 protein lacking globular domains 4–5 is secreted and incorporated into the Reichert’s membrane and embryonic basement membrane in these mutants [49]. Together, these results suggest that globular domains 4–5 of lama1 are required for the conversion of stem cells to polarized epithelium, but are not essential for the assembly of Reichert’s membrane and embryonic basement membrane in vivo.

Recently, a missense mutation in mouse lama1 gene (Y265C) has been found to contribute to the retinal vasculopathy, a disorder characterized by vitreal fibroplasia and vessel tortuosity [50, 51]. To further study the role of lama1 in this disease and bypass the embryonic lethal phenotype of lama1 null mice, a conditional knockout line with lama1 deficiency in epiblasts was generated by crossing the lama1 floxed (lama1^fl/fl) mice with the Sox2-Cre line [50]. These conditional knockouts are slightly lighter than the controls at birth but catch up by 8 weeks of age. They demonstrate diminished lama1 expression in the inner limiting membrane and exhibit similar but more severe retinal phenotype when compared to the lama1 Y265C mutants [50, 51], suggesting critical roles of lama1 in retinal development. In addition, these conditional knockout mice also show behavioral abnormalities and aberrant cerebellum formation [52], including decreased dendritic processes in Purkinje cells, disorganized Bergmann glial fibers and endfeet, and diminished proliferation and migration of granule cell precursors.

In addition to mice, zebrafish mutants have also been used to study the function of lama1. For example, lama1-knockdown zebrafish show degeneration of lens and reduction of eye size by 2 and 3 days postfertilization [53]. Additionally, axonal guidance defects, inner limiting membrane disruption, and shortened Muller cell endfeet are also observed in zebrafish with mutations in lama1 [54–56]. Furthermore, a zebrafish mutant lama1^a69/a69, which carries a cysteine-to-serine substitution at amino acid 56, displays shortened body axis and eye/len defects [57, 58]. These data suggest a critical role of lama1 in retinal development. However, it should be noted that these zebrafish mutants are larval lethal, which prevents the analysis of lama1’s function at later developmental stages.

Together, these data support an indispensable role of lama1 in embryonic development, and an important function of Sox2⁺ cell-derived α1-containing laminins in retina and cerebellum development/maturation. Although the functions of lama1 in other tissues/cell types remain elusive, the lama1^fl/fl line provides a valuable tool for these studies.

Laminin α2

Lama2 is predominantly expressed in striated muscles and peripheral nerves. Loss of function in this gene leads to aggressive muscle degeneration, known as muscular dystrophy [59–61]. The first studied mouse model for this disorder is dy/dy mice [62], which carries an unidentified mutation in lama2 gene [59, 63]. The dy/dy mice, which express low but detectable levels of lama2, develop muscle pathology at 3 weeks of age and usually die of unknown causes around 3–6 months. Another muscular dystrophy model involves a G-to-A mutation in the lama2 gene (named dy^2J), which results in abnormal splicing and generation of a truncated and non-functional lama2 protein [60]. Homozygous dy^2J (dy^2J/dy^2J) mice have a normal lifespan, but develop muscle weakness at about 3 weeks of age, which worsens progressively[60].

To further study the role of lama2, a lama2 null mouse line, termed dy^W/dy^W, was generated [64]. In these mice, under the endogenous lama2 promoter/enhancer elements, the lama2-coding sequence was replaced by lacZ sequence [64]. The dy^W/dy^W mice are passive, small, emaciated, and usually die between 2 and 4 weeks after birth [64]. They demonstrate muscle phenotypes similar to a disease known as congenital muscular dystrophy, including muscle fiber necrosis with regeneration, small myofibers with central nuclei, pronounced fibrosis, progressive lameness, and muscle paralysis [64]. In addition, another lama2 null mouse line (dy^3k/dy^3k) was generated by targeted deletion of amino acids 128–209 [65]. Like the dy^W/dy^W mice, lama2 expression and basement membrane structure are completely absent in the muscles of dy^3k/dy^3k mice [65]. These dy^3k/dy^3k mice show muscle pathology in early postnatal (P) stage (P9), deteriorate rapidly and aggressively, and die at 5 weeks of age [65], suggesting a more severe muscular dystrophic phenotype. All three lama2 mutant lines have been widely used as mouse models for congenital muscular dystrophy. In addition to the muscle phenotype, the dy^3k/dy^3k mice also demonstrate other phenotypes, including thinner and disrupted myelination [66], reduced motor conduction velocity in the sciatic nerve [66], compromise of blood brain barrier integrity [67], and smaller body size. Consistent with these reports, loss of lama2 has been found in human patients with congenital muscular dystrophy [61]; and brain abnormalities, including cortical anomaly, polymicrogyria, and abnormal white matter signals, have been reported in patients with lama2-deficient congenital muscular dystrophy [68]. Analyses of the dy^W/dy^W and dy^3k/dy^3k mice have significantly increased our understanding of the pathogenesis of congenital muscular dystrophy, and these mouse lines are two of the most widely used congenital muscular dystrophy models. These results suggest that lama2 exerts crucial functions primarily in skeletal muscles, nerves, and the CNS. It is worth noting that the smaller body size observed in dy^W/dy^W and dy^3k/dy^3k mice is not found in human patients. What causes this species-specific effect remains unclear. Answers to this question may lead to a novel congenital muscular dystrophy model that better replicates human pathology.

Due to structural similarity between lama1 and lama2, the therapeutic potential of lama1 in lama2-deficient mice was investigated extensively. Gawlik and colleagues generated a transgenic mouse line (dy^3kLNα1TG) that expresses lama1 in dy^3k/dy^3k background [69]. One line (No. 12) of these mutants shows strong lama1 expression in skeletal muscles and testes [69–72]. Compared to the dy^3k/dy^3k mice, these dy^3kLNα1TG mice (line No. 12) are bigger in size and have a normal lifespan [69, 72]. Muscle pathology (morphology and function) is improved to a remarkable degree in these mice. In addition, the aberrant distribution of integrin α7B and α- and β-dystroglycan in dy^3k/dy^3k mice is also reversed by the expression of lama1 in dy^3kLNα1TG mice [71]. Furthermore, loss-of-lama2-induced defects in testes, including abnormal timing in seminiferous tubule lumen formation, are partially rescued by the overexpression of lama1 [70]. In another line (No. 8) of the dy^3kLNα1TG mice, lama1 expression is detected in both skeletal muscles and the peripheral nervous system (PNS) [73]. Like line No. 12 described above, this line has a normal lifespan and body weight [73]. Additionally, the muscular dystrophy and peripheral nerve defects observed in dy^3k/dy^3k mice are dramatically reduced/corrected in this line (No. 8) of the dy^3kLNα1TG mice [73]. These studies clearly show that lama1 is able to rescue most of the phenotypes caused by loss of lama2, suggesting great therapeutic potential of lama1 in congenital muscular dystrophy. To further investigate the function of lama1 globular domains, a transgenic line (dy^3k/δE3) expressing lama1 lacking globular domains 4–5 in lama2 null background was generated [74]. Compared to the dy^3k/dy^3k mice, these dy^3k/δE3 mutants have prolonged lifespan and improved health [74]. Interestingly, diaphragm and heart muscle deficits, but not limb muscle defects are corrected by overexpression of the truncated lama1 [74]. Additionally, many unmyelinated axons and aberrant basement membranes in neuromuscular system are still observed in dy^3k/δE3 mice [74]. These results suggest that different muscles have different requirements for lama1 globular domains 4-5, and that these domains are essential for myelination in the PNS and important for basement membrane assembly.

Next, the therapeutic effect of laminin-111 protein was also tested in a variety of muscular dystrophy models. It has been shown that exogenous laminin-111 is able to reduce muscle pathology, enhance viability, improve muscle repair, and increase muscle strength in both dy^W/dy^W and mdx mice [75–78]. Furthermore, using a new muscular dystrophy model developed in our laboratory [79], we find that exogenous laminin is able to partially rescue muscle pathology at structural, biochemical, and functional levels [79]. Together, these data suggest that laminin-111 has therapeutic effect in muscular dystrophy and can be used to treatment this devastating disorder.

In addition to laminin, the therapeutic effect of agrin, another extracellular matrix protein, has been extensively studied. It has been shown that muscle-specific expression of a miniaturized form of agrin (miniagrin) in dy^W/dy^W and dy^3k/dy^3k mice significantly ameliorates muscle pathology and improves overall health [80, 81]. Late-onset expression of miniagrin remains beneficial, but to a lesser extent compared to early expression [82]. Similar result was reported in dy^W/dy^W mice administered with adeno-associated virus-expressing miniagrin [83]. Furthermore, expression of full-length agrin or a chimeric protein, in which the C-terminus of agrin is replaced by perlecan domain V, has been demonstrated to substantially slow down disease and lessen muscle pathology [82]. These data strongly suggest a therapeutic potential of agrin in lama2-deficient muscular dystrophy.

Besides extracellular matrix proteins, many other therapeutic options, including apoptosis inhibition, proteasome suppression, autophagy suppression, and fibrosis inhibition, have also been investigated. Genetic inactivation of Bax (proapoptotic protein) or overexpression of Bcl-2 (antiapoptotic protein) in dy^W/dy^W mice was able to partially ameliorate disease pathology [84]. Consistent with these results, pharmacological inhibition of apoptosis with Doxycycline or Omigapil in dy^W/dy^W mice partially amended muscle pathological changes [85, 86]. Proteasome inhibitors, MG-132 and Bortezomib, were able to elongate lifespan and diminish muscle pathology in dy^3k/dy^3k mice [87, 88]. Similar improvements in muscle pathology have been found in dy^3k/dy^3k mice after treatment with autophagy inhibitor 3-methyladenine [89]. Antifibrotic treatment with L-158809, a losartan derivative and angiotensin II receptor antagonist, led to reduced fibrosis and alleviated muscle pathology in dy^W/dy^W mice [90]. Together, these data suggest that apoptosis, proteasome, autophagy, and fibrosis can be targeted in the treatment of muscular dystrophy. However, it should be noted that these treatments are not as effective as targeting laminin or agrin.

Given that muscular dystrophy affects multiple signaling pathways and cellular functions, combinatorial approaches have been explored. For example, a cumulative effect was reported in dy^W/dy^W mice with both antiapoptosis (Bcl-2 overexpression or Omigapil application) and miniagrin treatments [91]. In addition, antiapoptosis (Bax inactivation) and proregeneration (muscle-specific IGF-1 overexpression) also demonstrated an additive beneficial effect in dy^W/dy^W mice [92]. The readers are referred to reference [93] for a systemic review of the treatments of lama2-deficient muscular dystrophy.

Laminin α3

Laminin-332 is expressed in various epithelial tissues [94, 95] and is linked to Herlitz junctional epidermolysis bullosa (H-JEB), a severe autosomal recessively inherited blistering skin disease that has no successful treatments so far [96, 97]. A C-to-T mutation, which results in a premature termination codon in lama3 gene, was identified in a human patient with H-JEB [97]. Next, a lama3 null mouse line was generated by removing exon A3, which causes a frame shift mutation and a premature stop codon that disrupt all possible α3-laminins [96]. The lama3 null mice are born at the Mendelian ratio, but develop progressive blistering in the skin after birth and die within 2–3 days [96]. Further studies show that these mutants develop skin phenotype similar to that in human H-JEB patients, including absence of lama3 expression in epidermal basement membrane, loss of anchorage function in the epidermis, abnormal hemidesmosomes, and defects in epithelial survival and ameloblast differentiation [96]. These results strongly suggest that the lama3 null mouse line is a valuable genetic model for H-JEB. Future research should focus on investigating the therapeutic potential of laminin-332 in H-JEB using lama3 null mice.

To further study the function of lama3, a lama3 floxed (lama3^fl/fl) line was generated by targeting the exon 42 of mouse lama3 gene [98]. By intratracheally treating these mice with adenovirus expressing Cre, Urich and colleagues reported that lung-specific loss of lama3 confers resistance to mechanical injury by increasing lung collagen expression [98]. In addition, resistance to mechanical ventilation and a mild inflammation were also found in these mice [98]. Together, these studies suggest fundamental roles of lama3 in skin epithelial development and lung function. The lama3^fl/fl mice will server as a valuable genetic tool and enable investigation of lama3’s roles in other tissues/organs.

Laminin α4

Lama4 is widely distributed in vascular basement membrane and basement membranes of other tissues, including skeletal muscle, nerves, heart, and kidney [99–102]. The lama4 null mice were generated by Thyboll and colleagues by targeting the promoter and exons 1 and 2 of lama4 gene [103]. The lama4 mutants are born at the expected Mendelian ratio, develop subcutaneous hemorrhage during embryonic and perinatal stages, and show anemia probably secondary to the internal bleeding [103], suggesting an important role of lama4 in vascular integrity at this stage. Interestingly, lama4 null mice surviving beyond the perinatal period have a lifespan comparable to the wild-type controls [103]. Consistent with these observations, defects in vascular basement membrane are observed in newborn but not adult (except the loss of lama4) lama4 null mice [103], suggesting compensation at later stages. Although most extracellular matrix proteins are not changed [103], lama5, which predominantly covers capillaries and postcapillary venules in wild-type brains [35], is expressed ubiquitously along the vascular tree in lama4 null mice [104]. In a multiple sclerosis model, lama4 null mice show substantially decreased disease susceptibility/severity owing to dramatically reduced T lymphocyte infiltration into the brain compared to wild-type mice [104], suggesting a permissive role of lama4 in T lymphocyte migration into the CNS. Further studies demonstrate that the ubiquitously expressed lama5 selectively inhibits integrin α6β1-mediated migration of T lymphocytes through lama4 into the brain in lama4 null mice [104]. These results suggest that compensatory expression of lama5 is also responsible for the less severe phenotype in lama4 null mice during multiple sclerosis.

Additional studies in adult lama4 null mice reveal defects in the neuromuscular junction, kidney, and adipose tissue. Patton and colleagues reported that the active zones and junctional folds are not precisely apposed to each other, although they form in normal numbers [105], suggesting that lama4 actively regulates the localization of synaptic specializations. Abrass and colleagues showed that these lama4 null mice have progressive fibrotic alterations in the kidney, including dilated microvessels, perivascular inflammation, tubulointerstitial fibrosis, and increased expression of collagen and fibronectin [106]. Moreover, the lama4 null mice also exhibit reduced weight gain in response to age and/or high fat diet, reduced epididymal adipose tissue mass, and altered lipogenesis [107], suggesting that lama4 modulates adipose tissue structure and function in a depot-specific manner. It is unclear, however, whether these phenotypes are caused by loss of lama4 directly or loss of lama4-induced lama5 expression indirectly. Elucidating this question would enrich our knowledge on laminin compensation and allow us to distinguish lama4’s function from lama5’s function.

To study the redundancy and compensation in the basal lamina of Schwann cells, which synthesize α2- and α4-containing laminins, two double mutant mouse lines (dy^2J/lama4^−/− and dy^3k/lama4^−/−) were generated. The dy^2J/lama4^−/− mice are smaller in size and rarely survive 3 months [108, 109]. Compared to either dy^2J/dy^2J or lama4^−/− mice, these double mutants demonstrate more severe phenotypes, including lack of myelination in peripheral nerves but not spinal roots and altered pancreatic acinar basement membrane composition [108, 109]. The dy^3k/lama4^−/− mice, on the other hand, are normal at birth but usually die within 2 weeks [108, 109]. These mutants show a nearly complete absence of sorting in both spinal roots and peripheral nerves [108], indicating a more substantial pathology. Like the dy^2J/lama4^−/− mice, the composition of pancreatic acinar basement membrane is affected in these dy^3k/lama4^−/− mutants [109]. These studies suggest that lama2 and lama4 do not fully compensate each other’s loss and they together coordinately regulate axonal ensheathment and Schwann cell function.

Laminin α5

Lama5 is expressed in virtually all basement membranes at early embryonic stage and then becomes restricted to specific basement membranes as development proceeds [37]. In mature vasculature, lama5 is predominantly found in capillary and postcapillary venule basement membranes [35]. To investigate lama5’s role in embryogenesis, Miner and colleagues generated lama5 null mice by targeting domains VI and V in the N-terminus of lama5 gene. The lama5 null embryos survive well past implantation stage, show apparent defects after E9, and die before E17 [37]. These lama5 mutants show defects in neural tube closure (exencephaly), digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth [37], indicating a critical role of lama5 in embryonic development. Consistent with these data, defects in neural crest migratory pathways and condensation into ganglia are also found in the lama5 null embryos [110], suggesting that lama5 is indispensable for proper migration and timely differentiation of some neural crest populations. Next, kidney defects characterized by abnormal glomerular basement membrane and glomerulogenesis are observed in these lama5 null embryos [111]. Some of mutants even lack one or two of the kidneys [111], indicating a crucial role of lama5 in kidney development. Lung defects manifested by incomplete lobar separation and absence of the visceral pleura basement membrane with normal lung branching morphogenesis and vasculogenesis are detected [112]. In addition, loss of lama5-induced defects in hair [113, 114] and intestine [115–117] are also reported. Fewer hair germs, failure of hair germ elongation, decreased length and structure of primary cilia at dermal papilla, as well as compromised expression of key morphogen/molecules are found in the skin of lama5 null mice [113, 114]. These lama5 mutants also show excessive folding of intestinal loops and delayed expression of specific markers in the intestine, suggesting an essential role of lama5 in intestinal smooth muscle development [117]. Mechanistic studies revealed abnormal Wnt and PI3Kinase signaling in the malformed intestine, linking these signaling pathways to the observed phenotype.

To further assess lama5’s function, a transgenic line that expresses a full-length lama5 transgene in lama5 null background (KO/Tg), was generated [118]. Transgene-derived lama5 successfully rescues the developmental defects of lama5 null mice and the KO/Tg mice are viable and fertile [118]. These KO/Tg mice, however, demonstrate very low level of lama5 expression in small intestine compared to wild-type controls [115]. This reduced lama5 expression in small intestine basement membrane is compensated by deposition of colonic laminin isoforms (lama1 and lama4), which leads to an architectural transformation from small intestinal mucosa to colonic mucosa [115]. To further study the specific function of lama5, two additional transgenic lines that ubiquitously express laminin α1/α5 chimeric proteins were generated. In one line, all five laminin-globular (LG) domains of lama5 are replaced by homologous sequences from lama1 in lama5 null background [118]. These transgenic mice die either before or shortly after birth, and show only limited improvement when compared to lama5 null embryos (reduced incidence of exencephaly) [118]. In the other transgenic line, the last three laminin-globular domains are replaced by homologous sequences from lama1 in lama5 null background [118, 119]. These transgenic mice are outwardly normal, live for several months, and die from nephrotic syndrome [118]. Additionally, they also demonstrate impaired postsynaptic but not presynaptic maturation at the neuromuscular junction [119]. These data suggest that lama5, especially its LG domains, has multiple functions and is essential for development.

Like the lama5 null mice, a hypomorphic mutation caused by the insertion of a PGKneo cassette in intron 21 of the lama5 gene leads to similar phenotypes [120]. This insertion causes aberrant splicing and exon skipping, resulting in dramatically reduced expression of lama5 [120]. Mice homozygous for this hypomorphic mutation show polycystic kidney disease, proteinuria, and death due to renal failure by 4 weeks of age [120], which can be rescued by expressing human lama5 transgene in podocytes [121]. These data again suggest an absolute requirement of lama5 in early kidney development.

Another lama5 mutant line that produces a nonfunctional lama5/βgeo fusion protein (lama5 trap mice), was generated by putting a trans-membrane domain and βgeo after the first 1763 amino acids of lama5 gene [43]. In both lama5 null and lama5 trap mice, discontinuous inner dental epithelial basement membrane, small tooth germ with defective cusp formation, decreased proliferation of dental epithelium, and defective enamel knot are observed [122], suggesting that lama5 is necessary for the proliferation and polarity of basal epithelial cells. In addition, severe defects in submandibular glands, including delayed epithelial clefting, reduced FGFR1b and FGFR2b expression, and disrupted epithelial cell organization and lumen formation, are found in both lama5 mutants [123], suggesting that lama5 actively regulates epithelial morphogenesis in submandibular glands.

To study the roles of lama5 at later developmental stages, a lama5 floxed (lama5^fl/fl) line was generated by inserting two loxP sites in the lama5 gene: one before exon 15 and the other after exon 21 [124]. By crossing the lama5^fl/fl line with lung epithelial cell-specific rtTA/tetO-Cre mice, an inducible lama5 conditional knockout mouse line was generated [124]. Mice exposed to doxycycline at E6.5 survive after birth and show severe defects in the lungs, including dilated/enlarged distal airspaces, perturbed distal epithelial cell differentiation, reduced alveolar type I and type II cells, increased apoptosis, decreased proliferation, and diminished capillary density [124], suggesting that epithelial cell-derived lama5 is necessary for alveolar epithelial cell differentiation and lung development. By utilizing podocyte-specific 2.5P-Cre, a conditional knockout mouse line with lama5 abrogation in podocytes was obtained [121]. These mutant mice are viable and show proteinuria, which deteriorates with age and eventually progresses to nephrotic syndrome [121], suggesting that podocyte-derived lama5 is crucial for the maintenance of glomerular filtration barrier integrity [121]. To study the effect of lama5 in neuromuscular junction maturation at postnatal stage, a conditional knockout line with muscle-specific deletion of lama5 was generated by crossing the lama5^fl/fl mice with human skeletal actin-driven Cre line [119]. These mutants are grossly normal with a normal lifespan, but demonstrate delayed pre- and post-synaptic maturation at the neuromuscular junction [119]. Furthermore, synaptic maturation is arrested in lama4/lama5 double mutants [119], suggesting that both lama4 and lama5 contribute to synaptic maturation at the neuromuscular junction. Altogether, these results suggest that lama5 has many important biological functions and the exact roles it plays depend on various factors, including tissue type, developmental stage, and compensation by other laminin α chains.

Laminin β1

Lamb1, a β subunit found in most laminin isoforms, is ubiquitously deposited in all basement membranes [42]. To investigate its function, a lamb1 trap line was generated [42, 43]. In these lamb1 trap mice, a gene trap vector is inserted in the 23rd intron of lamb1 to produce a fusion protein containing the first 1178 amino acids of lamb1 followed by a trans-membrane domain and βgeo [42, 43]. The lamb1 mutants die at E5.5 without the formation of either Reichert’s membrane or embryonic basement membrane [42]. In addition, no signs of cavitation and dramatically reduced trophoblast invasion are observed in these lamb1 mutants [42]. These defects are much worse than those in lama1 mutants and may explain the more severe phenotype of the lamb1 mutants. Recent study has identified a point mutation (T5460A) in lamb1 gene, which introduces a premature termination codon, leading to deletion of the last 57 amino acids from lamb1 chain [125]. Mice with this mutation exhibit dystonia-like hindlimb movements when awake and hyperextension when asleep [125], suggesting a pivotal role of the last 57 amino acids of lamb1 in CNS development and neuromuscular junction formation/maturation. This mouse line is a valuable tool for researchers studying movement disorders and neurological diseases.

Laminin β2

Lamb2 is widely expressed at the neuromuscular junction, kidney, and retina. To investigate its functional importance, a lamb2 null line was generated by targeting the second exon of lamb2 gene [126]. The lamb2 null mice are born at the expected Mendelian ratio and indistinguishable from their littermate controls at early postnatal stage [126]. Starting from P7, they become progressive weaker and die between P15 and P30 [126], suggesting that lamb2 is required for postnatal but not embryonic development. As expected, these lamb2 null mice demonstrate abnormal neuromuscular junctions. Structurally, the mutants show few active zones, evenly distributed vesicles in nerve terminals, abnormal Schwann cell processes, and few junctional folds in the postsynaptic membrane [126]. Functionally, significantly reduced miniature endplate potential frequency is observed in lamb2 mutants, although the passive membrane properties of the muscle fibers are normal [126].

In addition to the neuromuscular junction phenotype, a severe kidney defect, characterized by abnormal podocytes, increased glomerular filtration, and disorganized glomerular basement membrane, is observed [127–129]. To isolate the neuromuscular and kidney phenotypes, Miner and colleagues generated two transgenic mouse lines that express rat lamb2 in either muscle or podocytes under the lamb2 null background [128]. They elegantly show that deficiency of lamb2 in podocytes leads to disrupted glomerular filtration barrier, whereas loss of lamb2 in muscle is responsible for the abnormal synaptic architecture and failure to thrive [128].

Additionally, these lamb2 null mice also demonstrate retina defects, including abnormal outer segment elongation/electroretinograms/rod photoreceptor synapses, reduced tyrosine hydroxylase (TH) expression in type I TH neurons, decreased and increased density of type I and type II TH neurons, respectively [130, 131]. Whether retina-derived lamb2 is responsible for these defects, however, is unclear. Since lamb2 null phenotypes closely mimic the pathology of Pierson syndrome, which includes congenital nephrotic syndrome, ocular abnormalities, and muscular/neurological defects [132], these mice are used as an animal model for Pierson syndrome [128]. Consistent with these data, mutations in lamb2 gene have been found in human patients with Pierson syndrome [133, 134]. Based on these results, it is logical to hypothesize that adding lamb2 back would have therapeutic effect in Pierson syndrome. Future studies should focus on investigating the therapeutic potential of lamb2 in this disorder using lamb2 null mice.

Laminin β3

Lamb3 is a subunit of laminin-332, which actively participates in the pathogenesis of H-JEB. A mutation caused by insertion of an intracisternal-A particle (IAP) at an exon/intron junction, which disrupts the coding sequence of lamb3, has been identified in blistering (lamb3^IAP) mice [135]. Like lama3 null mice, these lamb3^IAP mice develop sub-epithelial blisters and abnormal hemidesmosomes lacking sub-basal dense plates, representing another mouse model of H-JEB [135]. Using this model, Muhle and colleagues demonstrated that prenatal delivery of lamb3 cDNA into the amniotic cavities of lamb3^IAP mice increases their lifespan slightly [136].

Recently, a point mutation in lamb3 (G628A), which is frequently observed in humans [12], was introduced into the mouse lamb3 gene. This mutation causes deletion of exon 7 and introduces a premature termination codon, most likely leading to a complete loss of lamb3 protein [137]. The homozygous G628A knockin mice show a phenotype very similar to the lamb3^IAP mice [137], suggesting that they can be used as an alternative mouse model of H-JEB. Future studies should focus on generating H-JEB models with longer lifespan, which would allow examination of these laminin chains at older ages. In addition, the therapeutic potential of laminin-332 in H-JEB should also be investigated in these lamb3 mutants.

Laminin β4

Lamb4 is a recently identified β chain. It is considered as a potential tumor suppressor that predisposes to colorectal cancer [138]. Consistent with this study, mutation in lamb4 and loss of lamb4 expression are found in some human colorectal cancer samples [139]. Currently, no lamb4 null mice are available.

Laminin γ1

Lamc1 is the most used γ chain and is found in the majority of laminin molecules [3, 4, 140]. The first lamc1 null mouse line was generated by homologous recombination targeting the first exon of lamc1 gene [141, 142]. These lamc1 mutants die at E5.5 due to severe developmental defects, including lack of basement membranes and failure of endoderm differentiation and parietal yolk sac development, suggesting that laminin γ1 chain is necessary for laminin assembly, endoderm differentiation, and early embryonic development [142]. Using these lamc1 mutants, Murray and Edgar further demonstrate that lamc1 regulates epiblast development and programmed cell death necessary for cavity formation [143]. Additionally, a lamc1 trap line that synthesizes a nonfunctional fusion protein containing the first 1091 amino acids of lamc1 followed by a trans-membrane domain and βgeo was also generated [43]. Like lamc1 null mice, these lamc1 trap mice die during embryogenesis at E5.5 [43], again indicating a critical role of lamc1 in early embryonic development. Recently, a lamc1 mutant line lacking the nidogen-binding site (lamc1III4^−/−) was generated by Willem and colleagues [29]. Approximately 40 % of the mutants die before E11.5, whereas 60 % of them die soon after birth [29]. These lamc1III4^−/− mutants show renal agenesis and impaired lung development due to locally restricted ruptures of basement membranes in the elongating Wolffian duct and alveolar sacculi, respectively [29]. These data suggest that interaction between nidogen and lamc1 is critical for early kidney morphogenesis and the formation of air–blood interface.

The early embryonic lethality of lamc1 null mice prevents investigation of lamc1’s functions at later stages. To overcome this limitation, a lamc1 floxed (lamc1^fl/fl) mouse line, in which exon 2 of the lamc1 gene was flanked by two loxP sites, was generated [144]. This lamc1^fl/fl line, by crossing with different promoter-driven Cre lines, allows abrogation of lamc1 expression in specific cell types, enabling loss-of-function studies of lamc1 in a cell type-specific manner.

By crossing this line with CaMKII-Cre, a conditional knockout line (CKO) with lamc1 deficiency in hippocampi, spinal cord, and peripheral nerves was generated [144]. These CKO mice show defects in neurite outgrowth and neuronal migration, including disrupted cortical layer structures and impaired axonal pathfinding, suggesting a critical role of lamc1 in brain development [145]. In additional to the CNS phenotype, these CKO mice also exhibit hindlimb paralysis and tremor due to disrupted lamc1 expression in peripheral nerves, which leads to failure in Schwann cell differentiation, axonal sorting and myelination [144].

By crossing the lamc1^fl/fl mice with P₀-Cre, a Schwann cell-specific lamc1 knockout line (P₀KO) was generated [146]. Similar to the CKO mice, these P₀KO mice develop tremor and progressive hindlimb paralysis, and usually die within 2 months [146]. Schwann cells in the mutants show reduced proliferation, increased apoptosis, and compromised extension of processes required for axonal sorting and axon-Schwann cell interaction [146], again suggesting that lamc1 is indispensable for Schwann cell survival, differentiation, and function.

Inactivating lamc1 gene in the developing mouse ureteric bud by crossing the lamc1^fl/fl mice with Hoxb7-Cre line leads to compromised renal collecting system growth and function [147]. These mice either fail to develop a ureteric bud or exhibit small kidneys with reduced proliferation/branching, abnormal renal vesicle formation, and water transport defect [147], suggesting γ1-containing laminins are required for normal kidney development and function.

By crossing the lamc1^fl/fl line with Nestin-Cre mice, a conditional knockout line (NKO) with lamc1 deficiency in neural progenitor cells (both neurons and glial cells) was generated [148, 149]. These NKO mice develop age-dependent and region-specific intracerebral hemorrhage [148] and blood–brain barrier breakdown in non-hemorrhagic mice [149]. By using the CKO mice and adenovirus expressing Cre under GFAP promoter, it has been demonstrated that these phenotypes are due to loss of γ1-containing laminins in astrocytes specifically [148, 149]. Further mechanistic studies reveal that loss of astrocytic laminin impairs vascular smooth muscle cell maturation [148] and pericyte differentiation [149], leading to intracerebral hemorrhage and blood–brain barrier breakdown, respectively.

Additionally, these NKO mice are unable to respond to angiotensin II (increase blood pressure), although they maintain normal baseline blood pressure [150]. In contrast to the NKO mice, mice with lamc1 deficiency in vascular smooth muscle cells specifically (SKO), generated by crossing the lamc1^fl/fl with SM22α-Cre mice, have a lower baseline blood pressure and reduced response to Angiotensin II [150]. These data suggest that nestin⁺ cell- and vascular smooth muscle cell-derived γ1-laminins have distinct functions, probably due to different laminin isoforms synthesized by these cells.

To assess the functional role of lamc1 in mural cells, a conditional knockout (PKO) line with lamc1 deficiency in PDGFRβ⁺ mural cells was generated by crossing the lamc1^fl/fl line with Pdgfrβ-Cre mice. These PKO mice are born at the expected Mendelian ratio, develop progressive muscle weakness soon after birth, and usually die within 4 months [79]. Further mechanistic studies demonstrate that PDGFRβ⁺ mural cell-derived γ1-containing laminins inhibit their proliferation, negatively regulate their adipogenic differentiation, and are required for their myogenic differentiation [79], suggesting a critical role of lamc1 in PDGFRβ⁺ mural cell stemness regulation.

Altogether, these studies suggest that lamc1 exerts a large variety of important functions, which are dependent on its cellular sources. The lamc1^fl/fl mouse line, which allows deletion of this gene in any cell type by crossing with cell type-specific promoter-driven Cre lines, provides a valuable tool for future studies.

Laminin γ2

Like lama3 and lamb3, mutations in lamc2 have also been identified in human patients with H-JEB [151]. To better study the functional role of lamc2, a lamc2 null mouse line was generated by targeted frameshift deletion of exon 8 of the lamc2 gene [152]. The lamc2 null mice are born at the expected Mendelian ratio, show blistering phenotype at P1–P2, and die by P5 [152], suggesting that lamc2 is not required for embryonic development. Poorly developed hemidesmosomes and induced apoptosis in the basal cells are observed in these mutants [152], replicating human H-JEB pathology. Additionally, a mild lung defect, characterized by abnormal tracheal hemidesmosomes but normal branching morphogenesis and epithelial differentiation, is found in these lamc2 mutants at P1–P2 [153], suggesting a minimal role of lamc2 in lung development through the saccular stage. Similarly, the therapeutic potential of laminin-332 in H-JEB should also be investigated in this mouse model.

To bypass the early lethality of lamc2 null mice and enable studies of lamc2’s function at later stages, a transgenic line expressing doxycycline-controllable human lamc2 gene under a keratinocyte-specific promoter on the lamc2 null background was generated [154]. These transgenic mice do not have skin blisters, are fertile, and survive over 1.5 years [154]. In these mice, human lamc2 co-localizes with mouse lama3 and lamb3, forming functional laminin-332, which rescues the alterations in the deposition of hemidesmosomal components, leading to restored formation of hemidesmosomes [154]. This transgenic line will be a valuable tool to study the functions of lamc2 in other organs and/or at later stages.

Laminin γ3

Lamc3 is widely but differentially expressed in a variety of tissues in mice. To investigate its biological function, a lamc3 null mouse line was generated by replacing exon 1 and part of the intron 1 of the lamc3 gene with a promoter-less IRES βgeo sequence [155]. Unlike other mutants, the lamc3 null mice develop a normal lifespan and show only minor abnormalities, including ectopic granule cells in the cerebellum and enhanced capillary branching in the outer retina [131, 155]. These data suggest that lamc3 may have some unique functions in the CNS and retina.

Both laminin β2- and γ3-containing laminins are present in the brain and retina during development. To further investigate their roles, a double knockout mouse line lacking both lamb2 and lamc3 (compound null) was generated by crossing lamb2 heterozygote with lamc3 null mice [131]. Like the lamb2 null mice, the compound null mice usually die within 4 weeks of age. In addition to fractured pial basement membrane, the compound null mice exhibit hallmarks of human cobblestone lissencephaly (type II), including laminar disruption, midline fusion, ectopic germinal zones, altered distribution of Cajal-Retzius cells, and abnormal radial glial cell morphology [156], suggesting that lamb2 and lamc3 are necessary for proper cortical lamination. In addition, these compound null mice also show defects in retina, the inner limiting membrane (ILM) in particular, at structural, biochemical, and functional levels. Besides the retinal defects observed in lamb2 null mice, the compound null mice also demonstrate disruptions in the laminar arrangement and ILM as well as alterations in Muller cell polarity and electrophysiology [131, 157, 158]. Many other features, including astrocyte migration and spatial distribution in the retina, retinal vascular development and maturation, and inwardly rectifying potassium channel and aquaporin-4 expression in Muller cells, are also affected in the compound null mice [158, 159]. Additionally, ERG recordings in response to flash stimuli delivered after prolonged dark adaption show that both the postsynaptic responses and photoreceptor-generated currents are severely disrupted in the compound null mice, whereas only the former is disrupted in lamb2 null mice [157]. Altogether, these data suggest that β2- and γ3-containing laminins are critical for the development of the CNS and retina.

Conflict of interest

The author declares no competing interests.