Tissue plasminogen activator (t-PA) is a secreted serine protease implicated in multiple aspects of development. In the adult rat brain, transcription of t-PA is an immediate-early response in the hippocampus following treatments that induce neuronal plasticity. To study the sequence elements that govern transcription of this gene, in situ analysis was used to define t-PA’s temporal and spatial expression pattern in midgestation embryos. Transgenic mice were then generated carrying t-PA 5′ flanking sequences linked to the E. coli lacZ gene. Constructs containing 4 kb of the flanking sequences (4.0TAMGAL) confer β-galactosidase activity mostly to the same tissues that exhibit high levels of t-PA mRNA by in situ analysis. In 4.0TAMGAL embryos from embryonic day 8.5 (E8.5) to 13.5 (E13.5), the majority of expression observed is localized to neural ectoderm-derived tissues. β-galactosidase activity is first detected in restricted neuromeres in the midbrain and diencephalon, at E8.5 and E9.5 respectively. At E10.5, transgene expression is observed in neural crest-derived cranial nerves and dorsal root ganglia, but not placode-derived cranial nerves. From E10.5 to E13.5, β-galactosidase activity is observed in postmitotic neurons of the midbrain, spinal cord, neural retina and the developing olfactory system. β-galactosidase activity is also detected in areas undergoing tissue remodeling such as the pinna of the ear, whisker follicles and the limbs. In adult mice, lacZ is expressed in the hippocampus and this expression was found to be enhanced upon seizure in the giant pyramidal neurons of CA3. These results reinforce the concept that t-PA plays a role in neurogenesis and morphogenesis, and identifies the promoter region that directs its transcriptional regulation both in development and in the CNS.

Vertebrate morphogenesis entails complex and precise interactions of cells with their environment, for which secreted proteolytic enzymes are likely to be important. The proteolytic system of plasminogen activators (PA) and plasminogen has been widely associated with migratory, remodeling and invasive events that occur in both normal and pathological conditions.

Plasminogen activators are specific serine proteases that catalyze the conversion of the zymogen plasminogen to plasmin. Plasmin can degrade a broad spectrum of extracellular proteins, as well as activate other proteases or latent growth factors. Two mammalian PAs are known to exist, tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). Although they catalyze the same reaction and are structurally similar, they are products of distinct genes and are often expressed in different tissues.

The PA/plasmin system has been connected with various processes throughout mammalian development (Pittman, 1990). With respect to the work presented here, it is notable that PAs have been implicated in neurogenesis (Menoud et al., 1989b; Pittman et al., 1989). Neural development is characterized by the migration of progenitor cells and the ability of neurons to extend processes (neurites) over long distances to specific targets. The PA/plasmin proteolytic cascade is believed to facilitate the migration of neurons and growing axons in culture systems and in neuronal regeneration experiments (Valinsky and Le Douarin, 1985; Moonen et al., 1982; McGuire and Seeds, 1990; Sallés et al., 1990). The involvement of PA in the nervous system is not limited to embryogenesis; t-PA activity is associated with specific regions of the adult mouse brain (Sappino et al., 1993) and induction of t-PA mRNA has been correlated in the rat with the neuronal plasticity linked to seizure, kindling and long term potentiation (Qian et al., 1993).

To study the possible functions of t-PA in mouse development, in situ analysis was used to define t-PA’s temporal and spatial expression pattern in midgestation embryos. Transgenic mice were then produced that carry a transgene with t-PA promoter sequences fused to the reporter gene, E. coli lacZ. β-galactosidase (β-gal) staining allowed analysis of transgene expression in embryos. Thus, these mice facilitated definition of the regulatory region(s) critical for appropriate spatial and temporal expression.

Transgenic mice carrying the lacZ gene under the control of 4 kb of 5′ flanking t-PA promoter (4.0TAMGAL) sequences have a pattern of tissue-restricted expression similar to that observed with the endogenous t-PA mRNA. These results demonstrate that 4.0 kb of the mouse t-PA promoter is sufficient in most tissues to confer faithful transgene expression. 4.0TAMGAL transgenic mice exhibit a discrete pattern of expression in derivatives of the neural ectoderm, such as in specific neuromeres of the diencephalon and mesencephalon. In addition, expression patterns correlate with neuronal differentiation in neural crest cells and the young neurons of the spinal cord. In the adult brain, the 4.0 kb promoter drives lacZ expression in the hippocampus and reproduces the immediate-early gene response observed previously (Qian et al., 1993) for t-PA after brief seizure events.

Generation of transgenic mice

Transgenic mice were generated (Hogan et al., 1986) using C57BL/6NTacfBR × DBA/2NTacfBR F1 (BDF-1) mice. t-PA 5′ reg-ulatory sequences fused to lacZ (Rickles et al., 1989) were excised from plasmid sequences using HindIII and BamHI restriction enzymes. The constructs were as described (Rickles et al., 1989) except the HindIII site at the t-PA promoter/lacZ junction was eliminated. The purified DNA was injected at a concentration of 2 ng/μl into the pronuclei of fertilized eggs isolated from superovulated females. Transgenic animals were identified by Southern analysis using a full-length lacZ probe. Comparison of the intensity of the lacZ hybridization to that observed for a t-PA 3′ UTR probe that detected only the endogenous gene was used to determine the number of copies of the transgene. PCR analysis was also performed on genomic DNA isolated from the tails of F0 embryos or F1 generation pups (Laird et al., 1991). The PCR primers for the lacZ gene were: sense primer, starting at nucleotide position 972 relative to the transcription start site: 5′ AGCAGAAGCCTGCGATGTC 3′; antisense primer, nucleotide position 1280: 5′ GTCAGACGATTCATTGGCAC 3′. t-PA primers were included within each sample as a PCR control (Richards et al., 1993).

Analysis of lacZ expression in the adult brain

β-gal activity was measured in the hippocampus from control transgenic mice and from transgenic mice in which seizure had been induced by intraperitoneal injection of pentylene tetrazol (PTZ) (50 mg/kg of body weight). Seizures began within 5 minutes of the injection. 4 hours after PTZ injection, the mice were subjected to an overdose of Avertin (0.02 ml/g of body weight) and perfused transcardially with 4% paraformaldehyde/0.2% glutaraldehyde in PBS. The tissue was dissected, fixed for an hour in fresh fixative solution, immersed overnight at 4°C in 30% sucrose in PBS and frozen in a dry ice/acetone bath.

Histological detection of β-gal activity

Typically, transgenic males were mated to non-transgenic BDF-1 females. Embryos were isolated at defined stages of development. The atlases of mouse development by Kaufman (1992) and Schambra et al. (1992) were used to identify structures and confirm embryonic stages. The morning of vaginal plug was considered E0.5. Embryos were prefixed, stained for β-gal activity and postfixed (Mendelsohn et al., 1991). For histological sections, embryos were dehydrated and embedded in paraffin. The paraffin blocks were cut into 6 μm sections (Frohman et al., 1990). Slides of sections were processed and counterstained with eosin. With the exception of nonspecific staining of amniotic tissue, non-transgenic embryos did not stain for β-galactosidase.

For adult brain, the tissue was subsequently sectioned (30 μm thickness) in a cryostat at −20°C. Sections were incubated with x-gal to determine sites of β-gal activity and (counter)stained with neutral red (Smeyne et al., 1992).

In situ hybridization

In situ hybridization on 6 μm paraffin sections of mouse embryos (Frohman et al., 1990) was performed using 33P-labelled antisense mRNA probes corresponding to either nucleotides 977-1409 of the t-PA coding region, or 1890-2401 of the t-PA 3′ untranslated region (Rickles et al., 1988). The two probes showed the same expression pattern for t-PA. No specific expression above background was detected when the corresponding sense probes were used.

RNase protection assay

RNA was prepared using the TRizol reagent (Gibco/BRL) from the hippocampus of adult transgenic mice which had or had not been subjected to PTZ-induced seizure. RNase protection experiments were performed using the RPA II kit (Ambion). A lacZ antisense transcript (410 nt) and a β-actin antisense transcript (360 nt, provided by the manufacturer) were used to hybridize to hippocampal RNA. The samples were analyzed on a 5% polyacrylamide-urea gel. Protected fragments of the predicted size (328 nt for lacZ, 250 nt for β-actin) were detected after hybridization and digestion of the RNA with the RNases. The intensity of the bands was quantitated and three separate experiments were averaged.

In vivo developmental analysis of the t-PA promoter

To investigate t-PA gene expression during development, we performed in situ analysis of tissue sections of embryos staged E9.5-E12.5 (discussed below, see Figs 1E, 2E, 3D,G, 4B-C). A summary of the expression data is provided here; relevant aspects of the expression patterns are described in detail in sub-sequent sections. At E10.5, t-PA mRNA expression was detected in the frontonasal process, in a ventral wall/floor plate region of the neural tube from the diencephalon to the caudal end of the spinal cord, in the caudal endoderm of the first, second and third branchial arches, in neural crest cells emanating from the spinal cord, in the dermomyotome and selected sclerotomal cells, in foregut and hindgut endoderm and associated mesoderm, in cardiac valve anlage, in the mesonephros and in the urogenital ridge (Figs 1E, 3D and data not shown). At E12.5, a generally similar pattern of expression was observed (data not shown). In addition, t-PA expression was detected in the olfactory epithelium and in retinal epithelium, in the optic nerve and at low levels in scattered mesodermal cells of neural crest origin adjacent to the forebrain, in spinal cord dorsal root ganglia and in the ventral motor region of the spinal cord (Fig. 3D,G and data not shown).

Fig. 1.

Temporal and spatial expression of the 4.0TAMGAL transgene in whole-mount X-Gal-stained mouse embryos (E9.5 to E13.5). (A) Lateral and (B) dorsal views at E8.75 demonstrate β-gal activity in the ms2 neuromere of the midbrain and branchial arches; (C) lateral view of E9.5 embryo with staining in the midbrain, hindbrain and cranial nerves; (D) lateral view at E10.5; (E) sagittal section showing t-PA mRNA expression in E10.5 embryo; (F) E11.5, and (G) E12.5 embryos; (H) dorsal view of E12.5; (I) lateral view of E13.5; (J) frontal view of E15.5 embryos. cm, core mesenchyme; drg, dorsal root ganglion; fp, floor plate; gam, gut-associated mesoderm; h, hindbrain; ht, heart; ms2, ms2 neuromere; n, nose; o, otic placode; sc; spinal cord; s, synencephalon; ur, urogenital ridge; trigeminal (V), facial (VII), glossopharyngeal (IX) and vagus (X) preganglion neural crest regions.

Fig. 1.

Temporal and spatial expression of the 4.0TAMGAL transgene in whole-mount X-Gal-stained mouse embryos (E9.5 to E13.5). (A) Lateral and (B) dorsal views at E8.75 demonstrate β-gal activity in the ms2 neuromere of the midbrain and branchial arches; (C) lateral view of E9.5 embryo with staining in the midbrain, hindbrain and cranial nerves; (D) lateral view at E10.5; (E) sagittal section showing t-PA mRNA expression in E10.5 embryo; (F) E11.5, and (G) E12.5 embryos; (H) dorsal view of E12.5; (I) lateral view of E13.5; (J) frontal view of E15.5 embryos. cm, core mesenchyme; drg, dorsal root ganglion; fp, floor plate; gam, gut-associated mesoderm; h, hindbrain; ht, heart; ms2, ms2 neuromere; n, nose; o, otic placode; sc; spinal cord; s, synencephalon; ur, urogenital ridge; trigeminal (V), facial (VII), glossopharyngeal (IX) and vagus (X) preganglion neural crest regions.

To attempt to recapitulate this mRNA expression pattern using lacZ as a reporter gene, we generated transgenic mouse lines carrying t-PA 5′-flanking sequence/lacZ fusion genes (Rickles et al., 1989). Initially, transgenic mice were generated with 4 kb (4.0TAMGAL) of t-PA 5′-flanking sequences fused to lacZ. This construct previously gave high levels of β-gal activity in transient cell culture (Rickles et al., 1989). Of eight transgenic mouse lines carrying 4.0TAMGAL, three lines (lines 62, 64 and 69) gave no detectable lacZ expression, four lines (lines 4, 40, 49 and 98) gave apparently identical patterns of lacZ expression and one line (line 72) did not significantly resemble the others. Transgene expression in line 72 may be influenced by genomic sequences near the site of integration. Within the four lines with apparently identical staining, the level of β-gal activity varied; however, the temporal and tissue expression patterns were consistent with the exception of the lacZ patterns in the eye (see below). The progeny of line 40 were used for the detailed description in this paper, because they expressed lacZ in higher levels. Non-transgenic embryos did not stain for β-gal except non-specifically in the umbilical vein and amniotic tissue (data not shown).

Previous analysis of t-PA promoter sequences in differentiated F9 embryonal carcinoma cells showed that as little as 250 bp of 5′ sequences conferred β-gal activity comparable to construct 4.0TAMGAL (Rickles et al., 1989). To analyze the in vivo activity of these sequences, transgenic mice were generated with constructs containing 500 bp (△1TAMGAL) or 250 bp (△11TAMGAL) of the t-PA promoter linked to lacZ. β-gal synthesis was not detected in F0 or F1 embryos (△1TAMGAL, 0/9 transgenic lines; △11TAMGAL, 0/11 transgenic lines). Therefore, sequences sufficient for transcriptional activity in differentiating F9 cells are not able to confer significant expression levels in vivo at E12.5. In the adult brain, expression of the △1TAMGAL transgene was observed in the hippocampus at very low levels, indicating that the 500 bp promoter sequences confer tissue specificity but lack activating elements required for higher expression.

t-PA expression during brain development

In vertebrates, the early embryonic brain can be divided into three regions: the forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon). By E9.5, the forebrain is further subdivided into the telencephalon and the more caudal diencephalon. The dorsal midbrain at this embryonic stage can be divided into two transverse neural segments or neuromeres, the rostral ms1 and caudal ms2 (Jacobson and Tam, 1982; referred to as neuromeres 4 and 5 in Sakai, 1987). lacZ expression is not detected in 4.0TAMGAL transgenic postimplantation embryos at E6.5, E7.5 or E8.0. Transgene expression is first detected at E8.5 in a restricted bilateral segment of neuroectoderm in the caudal region of the midbrain (Fig. 1A,B). Expression is confined to ms2 or possibly a subdomain within ms2. By E9.5, β-gal staining in the ms2 region has spread laterally and extends into the dorsal hindbrain (Fig. 1C). The β-gal staining likely represents cells of both neuroepithelium and neural crest origin. Many of these cells appear in tracks suggesting that they are migrating away from the ms2 neuromere staining band seen at E8.5. This expanding pattern is consistent with expression of the transgene in migrating neural crest cells which in mammals move in streams over many routes (Tan and Morriss-Kay, 1986; Serbedzija et al., 1992). Alternatively, it may represent de novo expression of the transgene in the expanded regions. Early development of the brain and spinal cord is characterized by a pseudostratified neuroepithelium consisting of proliferative cells. Upon leaving the cell cycle, the neural progenitor cells migrate into the periphery of the neuroepithelium which is termed the marginal layer. The postmitotic neurons of the marginal layer begin differentiation by initiating axonogenesis. The axon process grows through the brain to specific target cell(s) destinations. At E9.5 of brain development in 4.0TAMGAL embryos, β-gal staining in the neuroepithelium arises in the synencephalon (Fig. 1C), a neuromere of the diencephalon destined to become the interstitial nucleus of Cajal in the adult. The bilateral dorsal staining of the synencephalon is more pronounced by E10.5 (Fig.1D). The disperse β-gal staining in ms2 persists through E11.5 and staining is never observed in ms1. When examined by tissue sections, lacZ expression patterns of the synencephalon and ms2 appear different at E10.5 (Fig. 2A-C). The dorsal synencephalon contains β-gal-positive cells throughout the neuroepithelium, but the ms2 neuromere staining is restricted to postmitotic neurons at the periphery of the neuroepithelium. Embryos at E11.5 resemble E10.5 but with diminishing lacZ expression in synencephalon and the cranial nerves (Fig. 1F). By E12.5, β-gal activity is found in the dorsal neuroepithelium of ms2 containing the newest postmitotic neurons, and staining is also observed in the mesencephalic flexure. At this embryonic stage, β-gal activity in the synencephalon has dramatically decreased (Fig. 1G).

Fig. 2.

β-galactosidase activity in histological sections of the embryonic brain of 4.0TAMGAL transgenic embryos at E10.5. (A)Parasagittal section β-gal staining in the synencephalon of the diencephalon, the caudal midbrain and the hindbrain; (B) higher magnification view of the midbrain-hindbrain junction as shown in A; (C) medial transverse section through the synencephalon and the hindbrain, with β-gal activity throughout the neuroepithelium of the dorsal synencephalon and only peripheral in the ventral synencephalon; (D) coronal section displays lacZ expression in the trigeminal (V) (see also Fig. 1D) and facial (VII) preganglion neural crests and mantle region of the hindbrain containing postmitotic neurons; the acoustic ganglion (VIII) derived from the otic placode does not display β-gal activity; (E) transverse section at E10.5 through midbrain-hindbrain, showing mRNA hybridization in the floor of the hindbrain, with decreasing signal towards the dorsal side of the hindbrain. ba, branchial arches; d, diencephalon; h, hindbrain; m, midbrain; o,otic placode; s, synencephalon; t, telencephalon, d, dorsal, v, ventral.

Fig. 2.

β-galactosidase activity in histological sections of the embryonic brain of 4.0TAMGAL transgenic embryos at E10.5. (A)Parasagittal section β-gal staining in the synencephalon of the diencephalon, the caudal midbrain and the hindbrain; (B) higher magnification view of the midbrain-hindbrain junction as shown in A; (C) medial transverse section through the synencephalon and the hindbrain, with β-gal activity throughout the neuroepithelium of the dorsal synencephalon and only peripheral in the ventral synencephalon; (D) coronal section displays lacZ expression in the trigeminal (V) (see also Fig. 1D) and facial (VII) preganglion neural crests and mantle region of the hindbrain containing postmitotic neurons; the acoustic ganglion (VIII) derived from the otic placode does not display β-gal activity; (E) transverse section at E10.5 through midbrain-hindbrain, showing mRNA hybridization in the floor of the hindbrain, with decreasing signal towards the dorsal side of the hindbrain. ba, branchial arches; d, diencephalon; h, hindbrain; m, midbrain; o,otic placode; s, synencephalon; t, telencephalon, d, dorsal, v, ventral.

Fig. 3.

Developing spinal cord and dorsal root ganglia expression of TAM4.0GAL transgene mice in histological sections. (A) Coronal section at E10.5 through the spinal cord at the level of forelimbs (top) and hindlimbs (bottom). The spinal cord matures along a rostral-caudal axis; accordingly, β-gal activity is increased in the more mature ventral horns (mantle layer) than the caudal spinal cord; (B,C) high magnification view of the rostral and caudal spinal cord, respectively, as seen in A; (D) in situ hybridization of t-PA mRNA; a coronal section of an E10.5 embryo is shown. The same progression of development observed with the TAM4.0GAL mice is seen with mRNA along the rostro-caudal axis; (E) by E12.5 staining is seen in the ventral horn, intermediate neurons and dorsal horn of the spinal cord, as well as the dorsal root ganglion; (F) parasagittal section of E12.5. Staining is seen in the dorsal root ganglion and spinal cord; (G) expression of t-PA mRNA at higher levels in the dorsal side of E12.5 spinal cords along the motor neurons and dorsal root ganglia. c, caudal; dh, dorsal horn; drg, dorsal root ganglion; fp; floor plate; ht, heart; in, intermediate neurons; pv, prevertebrae; r, rostral; sc, spinal cord; sg, sympathetic ganglion; ur, urogenital ridge; vh, ventral horn.

Fig. 3.

Developing spinal cord and dorsal root ganglia expression of TAM4.0GAL transgene mice in histological sections. (A) Coronal section at E10.5 through the spinal cord at the level of forelimbs (top) and hindlimbs (bottom). The spinal cord matures along a rostral-caudal axis; accordingly, β-gal activity is increased in the more mature ventral horns (mantle layer) than the caudal spinal cord; (B,C) high magnification view of the rostral and caudal spinal cord, respectively, as seen in A; (D) in situ hybridization of t-PA mRNA; a coronal section of an E10.5 embryo is shown. The same progression of development observed with the TAM4.0GAL mice is seen with mRNA along the rostro-caudal axis; (E) by E12.5 staining is seen in the ventral horn, intermediate neurons and dorsal horn of the spinal cord, as well as the dorsal root ganglion; (F) parasagittal section of E12.5. Staining is seen in the dorsal root ganglion and spinal cord; (G) expression of t-PA mRNA at higher levels in the dorsal side of E12.5 spinal cords along the motor neurons and dorsal root ganglia. c, caudal; dh, dorsal horn; drg, dorsal root ganglion; fp; floor plate; ht, heart; in, intermediate neurons; pv, prevertebrae; r, rostral; sc, spinal cord; sg, sympathetic ganglion; ur, urogenital ridge; vh, ventral horn.

In the hindbrain, staining is first visualized in E10.5 embryos (Fig. 1D) where β-gal activity is restricted to individual cells at the periphery of the neuroepithelium lining (Figs 1D, 2A-C). β-gal activity remains high in the hindbrain from E10.5 to E13.5 (Fig. 1D,F-I).

t-PA expression in neural crest derivatives

Cranial sensory ganglia originate from the neural crest cells and cranial ectodermal placodes (Jacobsen, 1991). β-gal activity in 4.0TAMGAL embryos is detected in neural crest-derived cranial ganglia during midgestation (Figs 1C-D,F-G, 2D). At E9.5, β-gal staining was observed in the facial (the VII nerve) neural crest region (Fig. 1C). By E10.5 staining can be seen in all of the preganglion neural crest regions, which include the developing trigeminal (V), facio-(VII), glossopharyngeal (IX) and vagus (X) nerves (Fig. 1D). The otic placode-derived acoustic ganglion (VIII), which is not derived from neural crest, does not stain while the adjacent facial (VII) nerve contains β-gal activity (Fig. 2D). Although three of neural crest-derived ganglia (V, VII, IX) originate from specific rhombomeres of the hindbrain, r2, r4 and r6 respectively, the expression cannot be detected in the hindbrain at E9.5 when the rhombomere structures are present (Fig. 1C). After E9.5, the segmented bulges of the rhombomere pattern disappear.

The nerves and glial cells of the vertebrate peripheral nervous system (groups of neurons lying outside the brain or spinal cord) are neural crest-derived. Neural crest cells begin to migrate before neural tube closure. β-gal staining in 4.0TAMGAL transgenic embryos is detected in the neural crest cell-derived dorsal root ganglia cells (DRG), after they have migrated away from the neural tube. Staining can be detected in E10.5 embryos and become most intense at E12.5 (Fig. 1D,G,H). By E13.5, there is a noticeable decline in β-gal activity in the DRG (data not shown).

t-PA expression in the spinal cord

The adult spinal cord is polarized such that sensory neurons exist in the dorsal spinal cord and motor neurons lie in ventral spinal cord. In vertebrates, the spinal cord develops in two gradients, along the rostral-caudal and the dorsal-ventral axes. 4.0TAMGAL transgene expression closely follows the maturation of both of these axes. Neurogenesis in the murine spinal cord occurs along the ventral-dorsal axis where the neuroep-ithelium of the developing ventral spinal cord begins to differentiate and initiate axonogenesis of motor neurons at E10, progressing to the intermediate neurons about E11 and the sensory neurons of the dorsal region at E12 (Altman and Bayer, 1984; Wentworth, 1984a,b). β-gal staining becomes apparent in the ventral horn of mantle layer in the 4.0TAMGAL mice at E10.5 (Figs 1A, 3A-C). At this stage the presumptive motor neurons have migrated away from the neuroepithelium near the neural tube lumen and have begun to differentiate (Altman and Bayer, 1984).

Maturation of the spinal cord progresses from rostral to caudal with the cranial spinal cord more developed than its lumbar counterparts. At E10.5, β-gal activity is highest in the most cranial region and progressively declines in more caudal regions, suggesting that expression also follows the rostralcaudal axis (Figs 1D, 3A-C). By E11.5 staining remains in the motor neurons and has progressed into the maturing intermediate neuron region (data not shown). Sections of embryos at E12.5 have a decreased β-gal activity in the motor neurons and intermediate neurons. β-gal activity is observed in the sensory neurons at E12.5 (Fig. 3E,F). E13.5 embryos retain low but observable levels of expression of β-gal staining in the cervical, branchial and thoracic regions, but higher levels of β-gal activity in the more immature caudal regions (lumbar and sacral) (Fig. 1I and data not shown). Position and morphology identify the cells staining in the developing spinal cord as neurons.

t-PA expression in the developing eye, ear, nose, whiskers and limbs

In situ analysis was initially used to localize t-PA gene expression in the developing eye. t-PA mRNA was localized (E11.5-13.5) to the inner (neural) layer of the optic cup that represents cells destined to become the neural layer of the retina. Expression of 4.0TAMGAL in the developing eye was similarly observed in the inner layer, but also in regions in which t-PA mRNA was not found. This inconsistency suggests that 4 kb does not contain all the repressor regulatory elements required for localized expression within the eye. t-PA mRNA, as well as lacZ expression from 4.0TAMGAL, is observed in the olfactory epithelium at E12.5 (Fig. 4B,C).

Fig. 4.

t-PA expression in the developing olfactory system and eye. (A) Coronal section of the brain of 4.0TAMGAL transgenic embryos with β-gal staining in the nasal epithelium; (B) t-PA mRNA expression in the nasal neuroepithelium and (C) retinal neuroepithelium as detected by in situ analysis. All embryos at E12.5.

Fig. 4.

t-PA expression in the developing olfactory system and eye. (A) Coronal section of the brain of 4.0TAMGAL transgenic embryos with β-gal staining in the nasal epithelium; (B) t-PA mRNA expression in the nasal neuroepithelium and (C) retinal neuroepithelium as detected by in situ analysis. All embryos at E12.5.

lacZ expression is observed in the pinna of the ear at E12.5 and E13.5, stages associated with active evagination and tissue-remodeling (Fig. 1G). lacZ expression also is detected during development of the hair follicle of the snout at E13.5 (Fig. 1I). In this region, β-gal activity is observed in the epithelial cells, which represent the primordium of the vibrissae (sensory whiskers), and not in the surrounding mesenchyme cells. In addition, the primordium of the sinus hair follicles outside the snout express lacZ at E13.5 and E15.5 (Fig. 1I and data not shown). By E15.5, β-gal activity is observed in follicle epithelial cells that will give rise to body hair and β-gal activity continues in the vibrissae of the snout.

β-gal activity is first detected in the core mesenchyme of limb buds at E12.5 (Fig. 1G). By E13.5, core mesenchyme staining has increased and expanded; in addition, β-gal activity can be seen in interdigital mesenchymal regions (Figs 1I, 5A), which are sites associated with cellular necrosis (reviewed in Hinchcliffe, 1982). The developing forelimbs and hindlimbs of E15.5 embryos express lacZ in the distal tips of the medial four digits or the claw primordium, as well as the epithelium of the proximal region (Fig. 5B). There is a β-gal activity-free zone between the claw primordium and the proximal region of each digit. Lastly, the epithelium on the palmer surface express lacZ with the strongest expression in the area surrounding the prominent pads.

Fig. 5.

β-galactosidase activity in the forelimbs of 4.0TAMGAL transgenic embryos. (A) Dorsal view of E13.5 forelimb and (B) ventral view of E15.5 forelimb.

Fig. 5.

β-galactosidase activity in the forelimbs of 4.0TAMGAL transgenic embryos. (A) Dorsal view of E13.5 forelimb and (B) ventral view of E15.5 forelimb.

Expression of t-PA in the adult brain

Experiments in rat have shown that t-PA is an immediately-early gene expressed in response to events that involve neuronal plasticity (Qian et al. 1993), such as short seizures and kindling. Furthermore, Sappino et al. (1993), have detected t-PA activity in the adult hippocampus. The role that t-PA might play in this setting is not known. We therefore set out to investigate whether the 4.0 kb t-PA 5′ flanking fragment was sufficient to drive lacZ expression in the hippocampus and/or to respond to seizure.

Mice that have not been stimulated express lacZ in the hypothalamus, amygdala, cingulate gyrus, dentate gyrus, and CA1, CA2 and CA3 of the hippocampus, but not in CA4 (Fig. 6A, Ctr). We focused our attention primarily on the hippocampus because of its involvement in neuronal plasticity. After induction of seizure with PTZ, β-gal activity is observed at higher levels in all of the hippocampus regions in which β-gal had been observed previously, with the pyramidal cells of CA3 showing the highest level of induction (Fig. 6A, Exp). Despite the widespread induction, expression was still not detected in the CA4 area. Quantitation of the intensity of X-gal staining suggested that there had been a two-fold induction of β-gal activity in the CA3 hippocampal region after PTZ-seizure.

Fig. 6.

lacZ expression in the hippocampus of 4.0TAMGAL adult transgenic mice. (A) Expression of 4.0TAMGAL in untreated mice (Ctr) and after PTZ-induced seizure (Exp). 30 μm coronal tissue sections were prepared from control mice and from mice seized for 4 hours. After staining with X-gal, the images were converted to pseudocolor and the pixels in each of the hippocampal regions quantitated. The arrow indicates folded tissue which is not part of the hippocampal structures. CA1,CA3: hippocampal pyramidal fields; DG, dentate gyrus. (B) RNase protection assays on RNA from 4.0TAMGAL mice before and after PTZ-induced seizure. Total RNA (2.5 μg) extracted from the hippocampus of control (lane 1) or PTZ-treated mice (lane 2) was assayed simultaneously with antisense β-actin and lacZ transcripts (equal counts/minute for each probe). After analysis of the samples by 5% PAGE, the relevant bands were quantitated and the lacZ band normalized using β-actin. The lacZ bands appear less intense than those of β-actin because the 4.0TAMGAL transcript is less abundant than β-actin. Similar results were obtained in two additional experiments on different animals.

Fig. 6.

lacZ expression in the hippocampus of 4.0TAMGAL adult transgenic mice. (A) Expression of 4.0TAMGAL in untreated mice (Ctr) and after PTZ-induced seizure (Exp). 30 μm coronal tissue sections were prepared from control mice and from mice seized for 4 hours. After staining with X-gal, the images were converted to pseudocolor and the pixels in each of the hippocampal regions quantitated. The arrow indicates folded tissue which is not part of the hippocampal structures. CA1,CA3: hippocampal pyramidal fields; DG, dentate gyrus. (B) RNase protection assays on RNA from 4.0TAMGAL mice before and after PTZ-induced seizure. Total RNA (2.5 μg) extracted from the hippocampus of control (lane 1) or PTZ-treated mice (lane 2) was assayed simultaneously with antisense β-actin and lacZ transcripts (equal counts/minute for each probe). After analysis of the samples by 5% PAGE, the relevant bands were quantitated and the lacZ band normalized using β-actin. The lacZ bands appear less intense than those of β-actin because the 4.0TAMGAL transcript is less abundant than β-actin. Similar results were obtained in two additional experiments on different animals.

RNase protection experiments were used to quantitate the amount of lacZ RNA and demonstrated that there had been a four-fold increase in the amount of lacZ mRNA after PTZ-seizure (Fig. 6B). This induction is consistent with the 3-to 6-fold increase observed with the endogenous t-PA mRNA in the rat (Qian et al., 1993).

The results presented in this paper document the expression pattern of t-PA in the midgesta-tion mouse embryo and identify promoter elements that direct this pattern. The expression observed using in situ hybridization to localize t-PA mRNA is in most instances similar to the β-gal staining pattern generated in lacZ transgenic mice. Subtle differences in patterns that are generally similar might be attributable to altered stability of the chimeric lacZ mRNA and β-gal protein as compared to t-PA mRNA, or to differences in the translatability of the mRNAs, since the translation of t-PA mRNA is strikingly regulated during development (Huarte et al., 1987). β-gal protein is not observed in several tissues in which t-PA mRNA is expressed, most notably floor plate, cardiac valve anlage, gut endoderm and associated mesoderm, and the urogenital ridge. Presumably one or more positive and/or negative enhancer elements required for completely faithful expression of t-PA lie outside the 4 kb flanking region used for the current experiments. The activity of the t-PA promoter in neural ectoderm-derived tissues suggests that the protease functions in aspects of neurogenesis. The availability of a spatially functional promoter element raises the possibility of specifically perturbing processes in these tissues during development.

Previous studies have also analyzed plasminogen activators in vertebrate development. Sumi et al. (1992) examined t-PA and u-PA expression in the midgestation rat. They observed t-PA mRNA only in the floor plate at E12.5 during midgestation, but found high levels of u-PA in developing motor neurons and dorsal root ganglia. Menoud et al. (1989a) by in situ analysis detected patterns of t-PA mRNA expression in developing mouse embryos from E8 to E9.5 and found them very similar to those described in this paper.

Theuring et al. (personal communication) have used 3.1 kb of 5′ regulatory sequences from the human t-PA gene to drive expression of lacZ in transgenic mice. They observed β-gal activity in many of the same tissues as found with the mouse 4.0TAMGAL transgenics. Both the 4.0 kb mouse promoter and the 3.1 kb human promoter gave similar expression in the peripheral nervous system and several regions of the brain. However, a striking difference was observed in that neurons in the developing spinal cord did not express lacZ when fused to the human promoter. It is unclear whether this distinction is due to species specificity of the promoter sequences or differences in length of the promoter fragments. However, since the majority of the staining patterns are seen in both mouse and human t-PA/lacZ transgenic mice, it is clear that certain features of the promoters have been conserved in mammalian evolution.

The defined and varied pattern of t-PA expression in the developing mouse embryo suggests that the enzyme may serve several separate functions in development. For example, expression in regions of the diencephalon and midbrain is seen at E10.5 but the patterns within the developing neuroepithe-lium are different. In the diencephalon, lacZ is expressed throughout the pseudostratified neuroepithelium, whereas in the ms2 region of the midbrain, β-gal staining is restricted to the neuroblasts at the periphery of the neuroepithelium. We cannot deduce from the present studies if t-PA in brain neurogenesis has a singular function, or if there are distinct roles in various regions that lead to the discrete expression pattern.

Based on previous results and the t-PA expression pattern that we have observed, t-PA may function in numerous roles during midgestation development of the mouse. One prominent site of expression is in neural crest cells and their derivatives. Neural crest cells, which are derived from ectoderm, arise from interactions between the neural tube and the overlying ectoderm and then migrate along predetermined pathways to their final destinations (for reviews see Le Douarin and Smith, 1988; Bronner-Fraser, 1993); these migrations are invasive and ordered movements. Valinsky and Le Douarin (1985) have shown that migrating avian neural crest cells secrete high levels of u-PA (the only PA in avian systems), whereas surrounding tissues have low levels. Therefore, it is possible that t-PA activity facilitates cell migration. t-PA protease activity may be cell-surface bound since developing neuronal cells in culture and preimplantation mouse embryos can bind t-PA (Pittman et al., 1989; Verrall and Seeds, 1988; Carroll et al., 1993). In migrating monocytes, the u-PA receptor is localized to the leading edge of the cell allowing for directional movement (Estreicher et al., 1990). The expression of t-PA in neural crest cells might also function in growth factor activation. Brauer and Yee (1993) have demonstrated that cranial neural crest cells secrete latent TGF-β1 that can be activated by neural crest cell-generated plasmin.

t-PA is also expressed in areas undergoing extensive tissue remodeling. For example, the t-PA promoter is active during restructuring of the hindbrain (Fig. 1D,F-H), in the mesenchyme around the otic vesicle as it forms the pinna of the ear (Fig. 1I), in the whisker and sensory hair follicles as they evaginate at E13.5 (Fig. 1I,J), and the developing structures within the limbs (i.e., primordial claws and pads) (Fig. 5 and data not shown).

t-PA is also expressed in many necrotic tissues during development. In 4.0TAMGAL transgenic mice, t-PA expression is seen in the interdigital tissue webbing of the limbs at the time that these cells selectively die (Fig. 5). Also, neurons of the spinal cord and DRG, which also express t-PA, initially over-produce neurons of which as much as 50% will die during development (reviewed in Jacobsen, 1991). Thus, PAs may be involved in some aspect of apoptotic cell death or perhaps function to degrade material generated as a result of cell death. The functional significance of domains in the developing nervous system is beginning to be understood. In particular, there are defined regions early in development, such as neu-romeres, that have specific cell lineage capabilities and boundaries of expression (Hunt et al., 1991). As the molecular basis for specifying cell fate is becoming more clear, transcription factors such as homeobox genes and retinoic acid receptors (RARs) have emerged as crucial factors (McGinnis and Krumlauf, 1992). Several lines of evidence suggest that the t-PA gene might be a target for developmental transcription factors. The t-PA promoter is up-regulated by retinoic acid in F9 cells and contains at least 4 homeobox-binding motifs (Rickles et al., 1988; data not shown). t-PA expression in the limb bud appears similar to that of RAR-β2 expression whose expression can be regulated by retinoic acid in vivo (Mendelsohn et al., 1991). Furthermore, the patterns of expression of homeobox-containing genes En-1 and En-2 overlap that of t-PA in the ms2, midbrain-hindbrain junction and the hindbrain (Davis and Joyner, 1988; Davis et al., 1988). It will be interesting to examine t-PA gene expression further in transgenic mice with altered homeobox or RAR expression (i.e. by ectopic expression or gene disruption experiments).

Recent results suggest that t-PA is important in brain physiology. t-PA activity is found in discrete areas of the adult mouse brain, in particular, in the cerebellum, hippocampus and hypothalamus (Sappino et al., 1993). This expression may be regulated by a brain-specific factor that binds to the t-PA promoter in vitro (Pecorino et al., 1991). Moreover, transcription of the t-PA gene is induced in the rat hippocampus by various protocols (seizure, kindling, long term potentiation) that affect neuronal plasticity (Qian et al., 1993). Since all of these protocols induce structural changes in neurons, the role of t-PA in embryonic neurogenesis may be related to the mechanisms by which plasticity is maintained in the adult nervous system. Our data suggest that the 4 kb promoter fragment used to generate the transgenic mice described here is sufficient to recapitulate the transcriptional induction of t-PA after seizure. The role of t-PA in the adult brain and its function as an immediate-early gene are not yet understood. It has been speculated that t-PA may induce structural changes in neurons, either by activating a receptor, or by altering the interaction between proteases and protease inhibitors (Qian et al., 1993). Evidence was also provided to suggest that in addition to the three main PAIs known to exist in the adult rodent brain, other t-PA or plasmin inhibitors (Sappino et al., 1993) might be present.

It has been shown that brief epileptiform events induce the sprouting of mossy fibers (granular cell axons), which form de novo aberrant structures (bands) in the CA3 infrapyramidal layer (Ben-Ari and Represa, 1990). Sprouting of mossy fibers has been associated with the development of new synapses, but the mechanism by which they sprout is not known. t-PA has been implicated in neurite outgrowth and its rapid induction in the CA3 region might reflect the activation of a proteolytic activity required for mossy fiber sprouting.

We thank Melissa C. Colbert and Joel Levine for very helpful discussions, Kathy Dains, Dr. Robert Hitzemann and Barbara Hitzemann for help with the adult brain experiments and Yan-Ling Feng for producing the transgenic mice. We also appreciate the technical advice of David Colflesh and use of the University Microscope Imaging Center. S.E.T. was a recipient of an award from NIH post-doctoral training grant T32-DK07521 and a long-term fellowship from International Human Frontier Science Program (HFSPO). This work was supported by NIH grants (HD-17875) to S.S. and NIH (HD-29758) to M.A.F.

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